Proteins ingredients were prepared from regular tissue and tumor examples using AllPrep DNA/RNA/Proteins Mini Package (QIAGEN Ltd-UK, Crawley, UK)

Proteins ingredients were prepared from regular tissue and tumor examples using AllPrep DNA/RNA/Proteins Mini Package (QIAGEN Ltd-UK, Crawley, UK). in the mind, cardiac rhabdomyomas, and face angiofibromas. Mice heterozygous for At the ultimate end of treatment, animals had been humanely wiped out for evaluation of tumor burden and evaluation of proteins appearance and phosphorylation in regular tissue and tumor examples. AZD2014 (APExBIO, Houston, TX) at 4 mg/ml and rapamycin (LC Laboratories, Woburn, MA) at 1?mg/ml were prepared in automobile solution (2.5% PEG-400, 2.5% Tween-80, and 2.5% DMSO) respectively. Histology Evaluation of tumor burden in the kidneys of mice was performed as defined previously [8]. Mouse kidneys had been set in 10% buffered formalin saline for 24?hours, processed, and paraffin embedded. Six coronal parts of 5?m were prepared in a 200-m period from both kidneys of every mouse, stained with hematoxylin/eosin, and scanned using an Aperio program (http://www.aperio.com/?gclid = CNXN-8by4a UCFcINfAods3eg1w). Scanned pictures were employed for lesion quantification using ImageJ (http://rsbweb.nih.gov/ij). Lesion amount was driven, and optimum cross-sectional whole region including noncellular areas and cellular region of every renal lesion had been assessed. Tumor burdens had been estimated from entire areas and mobile regions of all lesions (cystic, papillary, and solid), cystic/papillary lesions, and solid carcinomas, respectively. The assessment was conducted regarding treatment status blindly. Immunohistochemistry (IHC) This research was accepted by the Institutional Review Plank from the Brigham and Women’s Medical center, Boston MA. Individual tumor mouse and areas kidney areas were prepared seeing that described above. Typical IHC was performed as defined [29] previously. Multiple sequential IHC (MS-IHC) was performed to colocalize multiple antigens in the same cells. An essential stage of MS-IHC was to totally strip previous principal antibodies to make sure performance and specificity of following principal antibody-antigen reactions. The process employed for stripping principal antibodies was improved from Kim et al. [30]. For MS-IHC, prior IHC-stained slides had been incubated in xylene for 10?a few minutes to eliminate coverslips and incubated in 50C within a buffered alternative containing 5% SDS, 0.5% mercaptoethanol, and 50?mm TrisCHCl (pH 7.5) for 60?a few minutes to strip principal antibodies, and lastly, the process was implemented for conventional IHC. SignalStain Increase Rabbit particular IHC Recognition Reagent (Cell Signaling Technology, Danvers, MA) and ImmPACT NovaRED Peroxidase Substrate or ImmPACT VIP Peroxidase (HRP) Substrate (Vector Laboratories, Peterborough, UK) had been utilized to stain antigens based on the package suppliers’ education. IHC or MS-IHC stained slides had been scanned to create digital slides for image catch using an Aperio program. Primary antibodies had been employed for IHC against phosphorylated S6 ribosomal proteins at S235/236, phosphorylated Akt at S473, E-cadherin, vimentin, FSP1, -SMA (Cell Signaling Technology, Danvers, MA), Ki67, and energetic caspase 3 (Abcam, Cambridge, UK). Traditional western blot Traditional western blot was performed as described [29] previously. Proteins extracts were ready from normal tissue and tumor examples using AllPrep DNA/RNA/Proteins Mini Package (QIAGEN Ltd-UK, Crawley, UK). Protein were purified based on the package supplier’s education. Twenty micrograms of proteins per test was separated on NuPAGE 4%-12% Bis-Tris Gels (Fisher Scientific UK Ltd., Loughborough, UK) and moved onto Amersham Protran Superior 0.2- or 0.45-m nitrocellulose blotting membranes (GE Healthcare UK Ltd., Small Chalfont, UK). Blots had been examined with ECL Select Traditional western Detection Package (GE Health care UK Ltd.), and indicators were discovered using Autochemi Imaging System (UVP, Upland, CA). Horseradish peroxidiseCconjugated secondary antibody against rabbit was utilized for Western blot (Cell Signaling Technology). Main antibodies were utilized for Western blot against phosphorylated S6 ribosomal protein at S235/236, 4E-BP1 at T37/46, Akt at S473, Akt at T308, and E-cadherin, vimentin, -actin (Cell Signaling Technology), phosphorylated PKC at T638 (Abcam); phosphorylated MDM2 at S166, mTOR at S2448, and mTOR at S2481 (Sigma-Aldrich, Dorset, UK). Statistical Analysis The Mann-Whitney test was used to compare tumor burden between treatment groups. Two-tailed Fisher’s exact test was used to compare protein expression in tumor cells obtained by IHC between treatment groups. and show co-expression of both epithelial and mesenchymal markers in tumor cells, suggesting partial EMT (15).To confirm this observation, MS-IHC was performed on the same kidney sections. Many hybrid epithelial-mesenchymal tumor cells were present as.Representative Western blots were presented to show phosphorylation of mTOR at S2448 and S2481, S6 at S235/236, Akt at T450 and S473, and PKC at T638. Open in a separate window Supplementary Physique 8 Expression of Ki67 and active caspase-3 in renal tumors of Tsc2+/? mice. Kidney sections were prepared from 16-month-old Tsc2+/? mice treated by vehicle, AZD2014, or rapamycin and stained by antibody against Ki67 and active caspase-3 with IHC. [4]. RCC occurs in about 4% of TSC patients and is characterized by diagnosis at a young age and pathological heterogeneity with clear-cell, papillary, and chromophobe carcinoma subtypes. TSC-associated lesions are also observed in other organs including lymphangioleiomyomatosis (LAM) affecting the lungs, subependymal giant cell astrocytomas in the brain, cardiac rhabdomyomas, and facial angiofibromas. Mice heterozygous for At the end of treatment, animals were humanely killed for assessment of tumor burden and analysis of protein expression and phosphorylation in normal tissues and tumor samples. AZD2014 (APExBIO, Houston, TX) at 4 mg/ml and rapamycin (LC Laboratories, Woburn, MA) at 1?mg/ml were prepared in vehicle solution (2.5% PEG-400, 2.5% Tween-80, and 2.5% DMSO) respectively. Histology Assessment of tumor burden in the kidneys of mice was performed as explained previously [8]. Mouse kidneys were fixed in 10% buffered formalin saline for 24?hours, processed, and paraffin embedded. Rabbit Polyclonal to OR10A7 Six coronal sections of 5?m were prepared at a 200-m interval from both kidneys of each mouse, stained with hematoxylin/eosin, and scanned using an Aperio system (http://www.aperio.com/?gclid = CNXN-8by4a UCFcINfAods3eg1w). Scanned images were utilized for lesion quantification using ImageJ (http://rsbweb.nih.gov/ij). Lesion number was decided, and maximum cross-sectional whole area including noncellular spaces and cellular area of each renal lesion were measured. Tumor burdens were estimated from whole areas and cellular areas of all lesions (cystic, papillary, and solid), cystic/papillary lesions, and solid carcinomas, respectively. The assessment was conducted blindly with respect to treatment status. Immunohistochemistry (IHC) This study was approved by the Institutional Review Table of the Brigham and Women’s Hospital, Boston MA. Human tumor sections and mouse kidney sections were prepared as explained above. Conventional IHC was performed as explained previously [29]. Multiple sequential IHC (MS-IHC) was performed to colocalize multiple antigens in the same cells. A crucial step of MS-IHC was to completely strip previous main antibodies to ensure efficiency and specificity of subsequent main antibody-antigen reactions. The protocol utilized for stripping main antibodies was altered from Kim et al. [30]. For MS-IHC, previous IHC-stained slides were incubated in xylene for 10?moments to remove coverslips and then incubated at 50C in a buffered answer containing 5% SDS, 0.5% mercaptoethanol, and 50?mm TrisCHCl (pH 7.5) for 60?moments to strip main antibodies, and SRT 1720 finally, the protocol was followed for conventional IHC. SignalStain Boost Rabbit specific IHC Detection Reagent (Cell Signaling Technology, Danvers, MA) and ImmPACT NovaRED Peroxidase Substrate or ImmPACT VIP Peroxidase (HRP) Substrate (Vector Laboratories, Peterborough, UK) were used to stain antigens according to the kit suppliers’ training. IHC or MS-IHC stained slides were scanned to generate virtual slides for photo capture using an Aperio system. Primary antibodies were used for IHC against phosphorylated S6 ribosomal protein at S235/236, phosphorylated Akt at S473, E-cadherin, vimentin, FSP1, -SMA (Cell Signaling Technology, Danvers, MA), Ki67, and active caspase 3 (Abcam, Cambridge, UK). Western blot Western blot was performed as described previously [29]. Protein extracts were prepared from normal tissues and tumor samples using AllPrep DNA/RNA/Protein Mini Kit (QIAGEN Ltd-UK, Crawley, UK). Proteins were purified according to the kit supplier’s instruction. Twenty micrograms of protein per sample was separated on NuPAGE 4%-12% Bis-Tris Gels (Fisher Scientific UK Ltd., Loughborough, UK) and transferred onto Amersham Protran Premium 0.2- or 0.45-m nitrocellulose blotting membranes (GE Healthcare UK Ltd., Little Chalfont, UK). Blots were analyzed with ECL Select Western Detection Kit (GE Healthcare UK Ltd.), and signals were detected using Autochemi Imaging System (UVP, Upland, CA). Horseradish peroxidiseCconjugated secondary antibody against rabbit was used for Western blot (Cell Signaling Technology). Primary antibodies were used for Western blot against phosphorylated S6 ribosomal protein at S235/236, 4E-BP1 at T37/46, Akt at S473, Akt at T308, and E-cadherin, vimentin, -actin (Cell Signaling Technology), phosphorylated PKC at T638 (Abcam); phosphorylated MDM2 at S166, mTOR at S2448, and mTOR at S2481 (Sigma-Aldrich, Dorset, UK). Statistical Analysis The Mann-Whitney test was used to compare tumor.SignalStain Boost Rabbit specific IHC Detection Reagent (Cell Signaling Technology, Danvers, MA) and ImmPACT NovaRED Peroxidase Substrate or ImmPACT VIP Peroxidase (HRP) Substrate (Vector Laboratories, Peterborough, UK) were used to stain antigens according to the kit suppliers’ instruction. lesions. Despite the fact that most AMLs are benign tumors, their propensity for spontaneous hemorrhage can have life-threatening consequences. Other TSC-associated kidney lesions include oncocytoma, malignant AML, and renal cell carcinoma (RCC) [2], [3], [4]. RCC occurs in about 4% of TSC patients and is characterized by diagnosis at a young age and pathological heterogeneity with clear-cell, papillary, and chromophobe carcinoma subtypes. TSC-associated lesions are also observed in other organs including lymphangioleiomyomatosis (LAM) affecting the lungs, subependymal giant cell astrocytomas in the brain, cardiac rhabdomyomas, and facial angiofibromas. Mice heterozygous for At the end of treatment, animals were humanely killed for assessment of tumor burden and analysis of protein expression and phosphorylation in normal tissues and tumor samples. AZD2014 (APExBIO, Houston, TX) at 4 mg/ml and rapamycin (LC Laboratories, Woburn, MA) at 1?mg/ml were prepared in vehicle solution (2.5% PEG-400, 2.5% Tween-80, and 2.5% DMSO) respectively. Histology Assessment of tumor burden in the kidneys of mice was performed as described previously [8]. Mouse kidneys were fixed in 10% buffered formalin saline for 24?hours, processed, and paraffin embedded. Six coronal sections of 5?m were prepared at a 200-m interval from both kidneys of each mouse, stained with hematoxylin/eosin, and scanned using an Aperio system (http://www.aperio.com/?gclid = CNXN-8by4a UCFcINfAods3eg1w). Scanned images were used for lesion quantification using ImageJ (http://rsbweb.nih.gov/ij). Lesion number was determined, and maximum cross-sectional whole area including noncellular spaces and cellular area of each renal lesion were measured. Tumor burdens were estimated from whole areas and cellular areas of all lesions (cystic, papillary, and solid), cystic/papillary lesions, and solid carcinomas, respectively. The assessment was conducted blindly with respect to treatment status. Immunohistochemistry (IHC) This study was approved by the Institutional Review Board of the Brigham and Women’s Hospital, Boston MA. Human tumor sections and mouse kidney sections were prepared as described above. Conventional IHC was performed as described previously [29]. Multiple sequential IHC (MS-IHC) was performed to colocalize multiple antigens in the same cells. A crucial step of MS-IHC was to completely strip previous primary antibodies to ensure efficiency and specificity of subsequent primary antibody-antigen reactions. The protocol used for stripping primary antibodies was modified from Kim et al. [30]. For MS-IHC, previous IHC-stained slides were incubated in xylene for 10?minutes to remove coverslips and then incubated at 50C in a buffered solution containing 5% SDS, 0.5% mercaptoethanol, and 50?mm TrisCHCl (pH 7.5) for 60?minutes to strip primary antibodies, and finally, the protocol was followed for conventional IHC. SignalStain Boost Rabbit specific IHC Detection Reagent (Cell Signaling Technology, Danvers, MA) and ImmPACT NovaRED Peroxidase Substrate or ImmPACT VIP Peroxidase (HRP) Substrate (Vector Laboratories, Peterborough, UK) had been utilized to stain antigens based on the package suppliers’ teaching. IHC or MS-IHC stained slides had been scanned to create digital slides for picture catch using an Aperio program. Primary antibodies had been useful for IHC against phosphorylated S6 ribosomal proteins at S235/236, phosphorylated Akt at S473, E-cadherin, vimentin, FSP1, -SMA (Cell Signaling Technology, Danvers, MA), Ki67, and energetic caspase 3 (Abcam, Cambridge, UK). Traditional western blot Traditional western blot was performed as referred to previously [29]. Proteins extracts were ready from normal cells and tumor examples using AllPrep DNA/RNA/Proteins Mini Package (QIAGEN Ltd-UK, Crawley, UK). Protein were purified based on the package supplier’s teaching. Twenty micrograms of proteins per test was separated on NuPAGE 4%-12% Bis-Tris Gels (Fisher Scientific UK Ltd., Loughborough, UK) and moved onto Amersham Protran High quality 0.2- or 0.45-m nitrocellulose blotting membranes (GE Healthcare UK Ltd., Small Chalfont, UK). Blots had been examined with ECL Select Traditional western Detection Package (GE Health care UK Ltd.), and indicators were recognized using Autochemi Imaging Program (UVP, Upland, CA). Horseradish peroxidiseCconjugated supplementary antibody against rabbit was useful for Traditional western blot (Cell Signaling Technology). Major antibodies were useful for Traditional western blot against phosphorylated S6 ribosomal proteins at S235/236, 4E-BP1 at T37/46, Akt at S473, Akt at T308, and E-cadherin, vimentin, -actin (Cell Signaling Technology), phosphorylated PKC at T638 (Abcam); phosphorylated MDM2 at S166, mTOR at S2448, and mTOR at S2481 (Sigma-Aldrich, Dorset, UK). Statistical Evaluation The Mann-Whitney check was utilized to evaluate tumor burden between treatment organizations. Two-tailed Fisher’s exact check was utilized to review proteins manifestation in tumor cells acquired by IHC between treatment organizations. and display co-expression of both epithelial and mesenchymal markers in tumor cells, recommending incomplete EMT (15).To verify this observation, MS-IHC was performed on a single kidney areas. Many cross epithelial-mesenchymal tumor cells.Five cystic/papillary lesions per pet were decided on and analyzed randomly.Supplementary Desk 2 Assessment of FSP1 Manifestation in Cystic/Papillary Lesions in Tsc2+/? Mice by IHC Evaluation (Two-Tailed Fisher’s Precise Check)IHC was performed to stain FSP1 as referred to in the Components and Strategies. lesions. Even though most AMLs are harmless tumors, their propensity for spontaneous hemorrhage can possess life-threatening consequences. Additional TSC-associated kidney lesions consist of oncocytoma, malignant AML, and renal cell carcinoma (RCC) [2], [3], [4]. RCC happens in about 4% of TSC individuals and is seen as a diagnosis at a age group and pathological heterogeneity with clear-cell, papillary, and chromophobe carcinoma subtypes. TSC-associated lesions will also be observed in additional organs including lymphangioleiomyomatosis (LAM) influencing the lungs, subependymal huge cell astrocytomas in the mind, cardiac rhabdomyomas, and cosmetic angiofibromas. Mice heterozygous for By the end of treatment, pets were humanely wiped out for evaluation of tumor burden and evaluation of proteins manifestation and phosphorylation in regular cells and tumor examples. AZD2014 (APExBIO, Houston, TX) at 4 mg/ml and rapamycin (LC Laboratories, Woburn, MA) at 1?mg/ml were prepared in automobile solution (2.5% PEG-400, 2.5% Tween-80, and 2.5% DMSO) respectively. Histology Evaluation of tumor burden in the kidneys of mice was performed as referred to previously [8]. Mouse kidneys had been set in 10% buffered formalin saline for 24?hours, processed, and paraffin embedded. Six coronal parts of 5?m were prepared in a 200-m period from both kidneys of every mouse, stained with hematoxylin/eosin, and scanned using an Aperio program (http://www.aperio.com/?gclid = CNXN-8by4a UCFcINfAods3eg1w). Scanned pictures were useful for lesion quantification using ImageJ (http://rsbweb.nih.gov/ij). Lesion quantity was established, and optimum cross-sectional whole region including noncellular areas and cellular region of every renal lesion had been assessed. Tumor burdens had been estimated from entire areas and mobile regions of all lesions (cystic, papillary, and solid), cystic/papillary lesions, and solid carcinomas, respectively. The evaluation was carried out blindly regarding treatment position. Immunohistochemistry (IHC) This research was authorized by the Institutional Review Panel from the Brigham and Women’s Medical center, Boston MA. Human being tumor areas and mouse kidney areas were ready as referred to above. Conventional IHC was performed as referred to previously [29]. Multiple sequential IHC (MS-IHC) was performed to colocalize multiple antigens in the same cells. An essential stage of MS-IHC was to totally strip previous major antibodies to make sure effectiveness and specificity of following major antibody-antigen reactions. The process useful for stripping major antibodies was revised from Kim et al. [30]. For MS-IHC, earlier IHC-stained slides were incubated in xylene for 10?moments to remove coverslips and then incubated at 50C inside a buffered answer containing 5% SDS, 0.5% mercaptoethanol, and 50?mm TrisCHCl (pH 7.5) for 60?moments to strip main antibodies, and finally, the protocol was adopted for conventional IHC. SignalStain Boost Rabbit specific IHC Detection Reagent (Cell Signaling Technology, Danvers, MA) and ImmPACT NovaRED Peroxidase Substrate or ImmPACT VIP Peroxidase (HRP) Substrate (Vector Laboratories, Peterborough, UK) were used to stain antigens according to the kit suppliers’ training. IHC or MS-IHC stained slides were scanned to generate virtual slides for picture capture using an Aperio system. Primary antibodies were utilized for IHC against phosphorylated S6 ribosomal protein at S235/236, phosphorylated Akt at S473, E-cadherin, vimentin, FSP1, -SMA (Cell Signaling Technology, Danvers, MA), Ki67, and active caspase 3 (Abcam, Cambridge, UK). Western blot Western blot was performed as explained previously [29]. Protein extracts were prepared from normal cells and tumor samples using AllPrep DNA/RNA/Protein Mini Kit (QIAGEN Ltd-UK, SRT 1720 Crawley, UK). Proteins were purified according to the kit supplier’s training. Twenty SRT 1720 micrograms of protein per sample was separated on NuPAGE 4%-12% Bis-Tris Gels (Fisher Scientific UK Ltd., Loughborough, UK) and transferred onto Amersham Protran High quality 0.2- or 0.45-m nitrocellulose blotting membranes (GE Healthcare UK Ltd., Little Chalfont, UK). Blots were analyzed with ECL Select Western Detection Kit (GE Healthcare UK Ltd.), and signals were recognized using Autochemi Imaging System (UVP, Upland, CA). Horseradish peroxidiseCconjugated secondary antibody against rabbit was utilized for Western blot (Cell Signaling Technology). Main antibodies were utilized for Western blot against phosphorylated S6 ribosomal protein at S235/236, 4E-BP1 at T37/46, Akt at S473, Akt at T308, and E-cadherin, vimentin, -actin (Cell Signaling Technology), phosphorylated PKC at T638 (Abcam); phosphorylated MDM2 at S166, mTOR at S2448, and mTOR at S2481 (Sigma-Aldrich, Dorset, UK). Statistical Analysis The Mann-Whitney test was used to compare tumor burden between treatment organizations. Two-tailed Fisher’s exact test was used to compare protein manifestation in tumor cells acquired by IHC between treatment organizations. and display co-expression of both epithelial and mesenchymal markers in tumor cells, suggesting partial EMT (15).To confirm.Scanned images were utilized for lesion quantification using ImageJ (http://rsbweb.nih.gov/ij). mind, cardiac rhabdomyomas, and facial angiofibromas. Mice heterozygous for At the end of treatment, animals were humanely killed for assessment of tumor burden and analysis of protein manifestation and phosphorylation in normal cells and tumor samples. AZD2014 (APExBIO, Houston, TX) at 4 mg/ml and rapamycin (LC Laboratories, Woburn, MA) at 1?mg/ml were prepared in vehicle solution (2.5% PEG-400, 2.5% Tween-80, and 2.5% DMSO) respectively. Histology Assessment of tumor burden in the kidneys of mice was performed as explained previously [8]. Mouse kidneys were fixed in 10% buffered formalin saline for 24?hours, processed, and paraffin embedded. Six coronal sections of 5?m were prepared at a 200-m interval from both kidneys of each mouse, stained with hematoxylin/eosin, and scanned using an Aperio system (http://www.aperio.com/?gclid = CNXN-8by4a UCFcINfAods3eg1w). Scanned images were utilized for lesion quantification using ImageJ (http://rsbweb.nih.gov/ij). Lesion quantity was identified, and maximum cross-sectional whole area including noncellular spaces and cellular area of each renal lesion were assessed. Tumor burdens had been estimated from entire areas and mobile regions of all lesions (cystic, papillary, and solid), cystic/papillary lesions, and solid carcinomas, respectively. The evaluation was executed blindly regarding treatment position. Immunohistochemistry (IHC) This research was accepted by the Institutional Review Panel from the Brigham and Women’s Medical center, Boston MA. Individual tumor areas and mouse kidney areas were ready as referred to above. Conventional IHC was performed as referred to previously [29]. Multiple sequential IHC (MS-IHC) was performed to colocalize multiple antigens in the same cells. An essential stage of MS-IHC was to totally strip previous major antibodies to make sure performance and specificity of following major antibody-antigen reactions. The process useful for stripping major antibodies was customized from Kim et al. [30]. For MS-IHC, prior IHC-stained slides had been incubated in xylene for 10?mins to eliminate coverslips and incubated in SRT 1720 50C within a buffered option containing 5% SDS, 0.5% mercaptoethanol, and 50?mm TrisCHCl (pH 7.5) for 60?mins to strip major antibodies, and lastly, the process was implemented for conventional IHC. SignalStain Increase Rabbit particular IHC Recognition Reagent (Cell Signaling Technology, Danvers, MA) and ImmPACT NovaRED Peroxidase Substrate or ImmPACT VIP Peroxidase (HRP) Substrate (Vector Laboratories, Peterborough, UK) had been utilized to stain antigens based on the package suppliers’ instructions. IHC or MS-IHC stained slides had been scanned to create digital slides for image catch using an Aperio program. Primary antibodies had been useful for IHC against phosphorylated S6 ribosomal proteins at S235/236, phosphorylated Akt at S473, E-cadherin, vimentin, FSP1, -SMA (Cell Signaling Technology, Danvers, MA), Ki67, and energetic caspase 3 (Abcam, Cambridge, UK). Traditional western blot Traditional western blot was performed as referred to previously [29]. Proteins extracts were ready from normal tissue and tumor examples using AllPrep DNA/RNA/Proteins Mini Package (QIAGEN Ltd-UK, Crawley, UK). Protein were purified based on the package supplier’s instructions. Twenty micrograms of proteins per test was separated on NuPAGE 4%-12% Bis-Tris Gels (Fisher Scientific UK Ltd., Loughborough, UK) and moved onto Amersham Protran Superior 0.2- or 0.45-m nitrocellulose blotting membranes (GE Healthcare UK Ltd., Small Chalfont, UK). Blots had been examined with ECL Select Traditional western Detection Package (GE Health care UK Ltd.), and indicators were discovered using Autochemi Imaging Program (UVP, Upland, CA). Horseradish peroxidiseCconjugated supplementary antibody against rabbit was useful for Traditional western blot (Cell Signaling Technology). Major antibodies were useful for Traditional western blot.