Figure 7

Specific inhibition of JNK2 decreases T172 phosphorylation of CDK4 in JNK1^−/− JNK2^MG/MG MEFs. From the cultures described in Figure 5, extracts of synchronized wild-type (wt), JNK1^−/−, and JNK1^−/− JNK2^MG/MG MEFs stimulated with FBS for 24 h in the presence or absence of 3MB-PP1, SP600125 or JNK-IN-8 were Ip with anti-cyclin D1, and separated by 2D-gel electrophoresis. Immunodetection was done with antibodies directed against CDK4 or its T172 phosphorylation (p-CDK4(T172)). Chemiluminescence images of blots were captured with a Vilber-Lourmat Solo7S camera and quantified using the Bio1D software (Vilber-Lourmat). The coincidence of the detections is shown by their superposition after conversion to green or red (respectively) using Photoshop software (merged, Adobe Systems Inc., San Jose, CA, USA). The percentage of the T172-phosphorylated form of CDK4 (arrows) versus the sum of Ip CDK4 forms is indicated (% P-CDK4) and shown in the bar graphs. Results with the three inhibitors were reproduced in independent experiments three times in wt MEFs and twice in JNK1^−/− JNK2^MG/MG MEFs.