Supplementary Figure 5 : ROS-induced PEX5 ubiquitination is ATM-dependent.

From: ATM functions at the peroxisome to induce pexophagy in response to ROS

Supplementary Figure 5

(a) HEK293 cells expressing Myc-PEX5-WT or PEX5 mutants (K28R, K52R, K170R, K204R, K209R, K292R) and HA-Ub constructs were co-immunoprecipitated using anti-HA and blotting with an anti-Myc antibody. Input lysates were immunoblotted using the indicated antibodies. (b) WT (GM08399) and AT (GM05849) fibroblasts were treated with H2O2 for 6 h and immunoprecipitated with anti-PEX5 and immunoblotted with anti-Ub antibodies. Input lysates were immunoblotted using the indicated antibodies. (c) HEK293 cells transfected with Myc-PEX5-WT and HA-Ub for 24 h following a prior siRNA knockdown of ATM for 48 h, were treated with H2O2 for 6 h and immunoprecipitated with anti-HA and immunoblotted with anti-Myc antibodies. Input lysates were immunoblotted using the indicated antibodies. (d) HEK 293 cells were transfected with SiRNA for PEX2, PEX10 and PEX12, and treated with H2O2 for 6 h. Immunoprecipitation was performed with an anti-PEX5 antibody followed by immunoblotting for endogenous ubiqintin. RT- PCR was performed to confirm siRNA knockdown of PEX5, PEX10 and PEX12. (e) HEK293 cells transfected with Myc-PEX5-WT/Myc-PEX5-K209R and HA-Ub constructs for 24 h following a prior siRNA knockdown of PEX2/10/12 for 48 h were immunoprecipitated with anti-HA and immunoblotted with anti-Myc antibodies. Input lysates were immunoblotted using the indicated antibodies. Uncropped images of western blots are shown in Supplementary Fig. 9.