Fig. 3.
Generation and characterization of the inactive p38γ knock-in mouse. (A) Diagram showing the endogenous p38γ gene, the targeting construct vector generated, the targeted allele with the neomycin selection cassette still present (Neo), and the targeted allele with the neomycin cassette removed by Cre recombinase. The grey boxes represent exons and the black triangles, the LoxP sites. The knock-in allele with the D171A mutation in exon 7 is marked (dark grey). Genomic DNA purified from tail biopsy sample was used as a template for PCR, resolved on a 1% agarose gel and examined by ethidium bromide staining. (B) Lysates from WT, p38γ−/− or p38γ171A/171A MEFs (30-50 μg of protein) were immunoblotted with specific antibodies for each protein indicated. (C) WT or p38γ171A/171A MEFs were exposed to 0.5 M sorbitol (15 minutes) and activation of the indicated MAPK was examined using phospho-specific antibodies. To analyse activation of p38γ, this isoform was immunoprecipitated with anti-p38γ antibody from 2 mg cell lysates and immunoblotted with the p38 phospho-specific antibody. (D) WT, p38γ171A/171A or p38γ/δ−/− MEFs were treated as in C and endogenous hDlg immunoprecipitated with anti-hDlg antibody from 0.5 mg protein lysate. Pellets were immunoblotted with anti-P-hDlgS158 or anti-hDlg.