Supplementary Figure 4: Characterization of AMBRA1NT-PX and AMBRA1CT-PXP mutants.

(a) HEK293 cells were transfected with vectors encoding for AMBRA1WT and AMBRA1NT-PXP. Protein extracts were immunoprecipitated using an anti-PP2A-C antibody and the immunocomplexes were analysed by western blot, using an anti-AMBRA1 and anti-PP2A-C antibodies. (b) HEK293 cells were transfected with vectors encoding for AMBRA1WT and AMBRA1CT-PXP. Protein extracts were treated as in a. (c) HeLa cells, transfected with βGal, as a control, AMBRA1WT or AMBRA1PXP, were lysed and proteins extracts were analysed for p62 levels and LC3I to LC3II conversion by western blot analysis. (d) HEK293 cells were transfected with vectors encoding for AMBRA1WT and AMBRA1PXP. Endogenous BECLIN 1 was immunoprecipitated from protein extracts and the immunocomplexes were analysed by western blot, using antibodies against BECLIN 1 and AMBRA1. (e) HEK293 cells were transfected as in d. Endogenous ULK1 was immunoprecipitated from protein extracts and the immunocomplexes were analysed by western blot, using antibodies against ULK1 and AMBRA1. The vertical line represents a splice mark of samples on the same gel. The brackets indicate bands corresponding to over-expressed AMBRA1.