Figure 4: Colocalization of VANGL2 and p62/SQSTM1 in late endosomes.

(a) Immunofluorescence staining of SKBR7 cells showed colocalization of endogenous VANGL2 (green) and p62/SQSTM1 (red) in discrete cytoplasmic puncta. Scale bar, 10 μm. The mean Pearson correlation for VANGL2 and p62/SQSTM1 is 0.62, calculated using the Image J software for ∼15 cells per field of view and from 10 images. (b) Partial colocalization of VANGL2 and p62/SQSTM1 in late endosomes of SKBR7 cells stained with the LAMP1 marker. Scale bar, 20 μm. (c) SKBR7 ells were cultured in a nutrient-deprived medium and treated with 100 nM bafilomycin A1 (6 h) before fixation. Double labelling against VANGL2 (arrowheads) and p62/SQSTM1 (arrows), as described in Methods, showed accumulations of both markers in vesicular structures probably resembling endosomes/amphisomes. Scale bar, 200 nm. (d) IMCD3 cells were treated with PBS or EGTA (5 mM) for 30 min. Immunofluorescence and confocal analysis were performed using the indicated antibodies. Scale bar, 10 μm. Inserts show colocalized VANGL2 and p62/SQSTM1. (e) The VANGL2–p62/SQSTM1 complex was recovered in confluent SKBR7 or IMCD3 cells with 2G4 mAb (IP VANGL2) but not a control antibody (IP IgG2a ctrl) as seen using western blot analysis with the indicated antibodies. The complex was more abundant in cancer cells (SKBR7) than in polarized cells (IMCD3). Note that human (SKBR7 cells) and murine (IMCD3 cells) p62/SQSTM1 run at different molecular weights. (f) Lysates of confluent IMCD3 cells treated for 30 min with PBS or EGTA were subjected to 2G4 mAb immunoprecipitation (IP VANGL2). Immunoprecipitated proteins were probed by western blot analysis with the indicated antibodies. Increased amounts of VANGL2–p62/SQSTM1 complexes were recovered after EGTA treatment.