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Figure 5. The VANGL2–p62/SQSTM1 complex regulates JNK phosphorylation.(a) Downregulation of VANGL2 in SUM149 cells using a specific shRNA led to reduced JNK phosphorylation induced by Wnt5a (100 ng ml−1 for the indicated times). JNK is represented by two isoforms: p54 and p46. Wnt5a led to p46 (phospho-p46) and not p54 (phospho-54) phosphorylation. Relative quantification of immunoblots (phosphorylated JNK/total JNK) is representative from three independent experiments and use of two different shRNAs. (b) Expression of GFP-p62DN, but not GFP, in SUM149 cells led to decreased p46 JNK phosphorylation (phospho-46) induced by 100 ng ml−1 of Wnt5a at the indicated times. Relative quantification of immunoblots (phosphorylated JNK/total JNK) is representative from three independent experiments. (c) SUM149 cell extracts were added with the control peptide (Ctrl peptide) or the p62/SQSTM1 peptide (p62DN) that inhibited recruitment of JNK and p62/SQSTM1 to VANGL2. (d) Proteins extracted from SUM149 cells treated or not with serum were immunoprecipitated with anti-VANGL2 antibody and blotted with the indicated antibodies. p62/SQSTM1, JNK and phosphorylated JNK (phospho-p54 and phospho-p46) were present in the VANGL2 complex. Larger Image Printer Friendly View

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