The inactive form of GSK3- which is phosphorylated on serine ?9 was elevated in the spinal cord of P35 KO mice (Fig

The inactive form of GSK3- which is phosphorylated on serine ?9 was elevated in the spinal cord of P35 KO mice (Fig. and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked. and in vivo. Open in a separate Tenapanor window Physique 4 Phosphorylation of NF-H and NF-H tail domain name fusion proteins by proline directed kinases(A) Both cdk5 and ERK 1,2 phosphorylate recombinant NF-H and and generate the RT-97 phosphoepitope. Western blot analysis of full length bacterially expressed NF-H immunostained with RT-97 after it was phosphorylated using the immunoprecipitate (IP) of cdk5 as enzyme source (lane 2) and ERK 1,2 (lane 1) and lane 3 represents a 14 mer KSPXK repeat fusion protein derived from human NF-H tail sequence, immunostained with RT-97 after phosphorylation using immunoprecipitate of ERK1,2 and IP of cdk5 (lane 5) and lane 4 represents the fusion protein alone. (B) ERK2 phosphorylation KSPXXXK fusion protein elicits RT-97 IR: 10 g of 24 KSPXXXK repeat fusion protein was phosphorylated with a mixture of recombinant ERK2 and Mek1 in a time course reaction ranging from 0C2 hours (0, 0.5, 1, 1.5 and 2 hrs represented in lanes 1 through 5 respectively) in triplicate. Equal aliquots were removed from the reaction combination and were electrophoresed on 7% SDS gels followed by electrotransfer onto nitrocellulose membrane in order to immunostain with RT-97 monoclonal antibody (lower panel). One gel was stained with silver to discern the protein (top panel) dried and subjected to autoradiography to visualize phosphorylation (middle panel). Inhibition of ERK 1,2, JNK1,2 or cdk5 alter RT-97 immunoreactivity in hippocampal neurons To investigate the regulation of the RT-97 phospho epitope Western blot analysis was carried out on whole homogenates of four units of spinal cord from mice at postnatal days 1, 3, 5 and 15 using RT-97 monoclonal antibody and polyclonal antibodies to the neurofilament kinases, phospho and total ERK 1,2, JNK 1,2 , cdk5. Calnexin was measured to confirm equivalent protein loading. Blots were developed using an HRP based chemiluminescence (ECL kit from Amersham) B and C: The films were scanned using HP Scan Jet 4070 and band densities were measured using NIH image J 1.37 V software, and analyzed using Graph Pad Prism-4 software. DCH: Western blot analysis was carried out on whole homogenates of three units of spinal cord from p-35 knock out and control mice using monoclonal antibodies to phospho-neurofilament H and polyclonal antibodies to neurofilament kinases ERK 1,2 and cdk5/p-35. Blots were developed using an HRP based chemiluminescence (ECL kit from Amersham) E: The band densities offered in the bar graph were measured using NIH Image J 1.3 V software after scanning the films using HP Scan. Jet 4070. F: Western blot analysis of samples used in physique 9 D, using polyclonal antibodies to inactive GSK3- (phosphorylated at serine 9) and total GSK3-. G: The band densities offered in the bar graph were measured using NIH Image J 1.3 V after scanning the films using HP Scanjet 4070. H: Western blot analysis of whole homogenates of brain from four units of E-16.5 cdk5 knock out and control mouse FAXF embryos, using polyclonal antibodies against cdk5, P-ERK 1,2, ERK 1,2, P-JNK 1,2 and JNK 1,2. Given that cdk5 inhibition only modestly lowered RT-97 IR levels in neurons, we analyzed its possible role further by investigating mice lacking P35 (P35 Tenapanor KO), the protein activator of cdk5. The cdk5 activity of brain Tenapanor and spinal cord from P35 knockout mice was 10C20 % of wild type mice, suggesting that P35 is the major activator of cdk5 in these tissues (Ohshima et al. 2001). Despite.