Figure 3: Identification of the signalling adapter p62/SQSTM1 as a direct binding partner of VANGL2.
(a) Endogenous interaction between VANGL2 and p62/SQSTM1 is revealed by western blot analysis after immunoprecipitation using SKBR7 cell extracts. TL is total lysate. Crtl ab is an isotype-matched antibody. (b) The VANGL2–p62/SQSTM1 interaction occurs independently of LC3. Proteins were extracted from SUM149 cells and co-immunoprecipitations were carried out with the indicated antibodies. LC3 co-precipitates with p62/SQSTM1 but not with VANGL2. Reciprocally, VANGL2 co-immunoprecipitates with p62/SQSTM1 but not with LC3. IP control antibodies (IP crtl ab) are a polyclonal rabbit (for IP LC3) and a monoclonal rat antibody (for IP VANGL2). IgHs are immunoglobulin heavy chains. (c) GST pulldown assays of in vitro translated GFP–VANGL2 (full length: WT, N-terminal 1–102: NT, C-terminal 242–521: CT) showed that VANGL2 WT and CT directly bind to GST–p62/SQSTM1 (GST-p62) but not to GST. Asterisks indicate in vitro translated VANGL2 (top panel) and GST (bottom panel) proteins. AR, autoradiography; CBB, Coomassie Brilliant Blue. (d) A p62/SQSTM1 peptide (p62DN) disrupts the endogenous VANGL2–p62/SQSTM1 complex. SUM149 cell protein extracts were incubated with the indicated peptides p62DN or scrambled control peptide (Ctrl peptide) at 100 μM. VANGL2 was then immunoprecipitated (IP VANGL2) and bound proteins were immunoblotted with the indicated antibodies. TLs showed that equal amounts of proteins were present in each condition.
