Figure 8.

The indispensable role of VDAC1 in the mitophagy inhibition by TSPO. (A) Cell lysates from WT MEFs were immunoprecipitated with anti-TSPO antibodies and associated VDAC1 was detected with anti-VDAC1 antibodies by western blotting; VDAC1 co-immunoprecipitates with TSPO. Five% of the lysate used for the immunoprecipitations was loaded for the input and probed with anti-VDAC1 (illustrating VDAC1 input) and anti-GAPDH (illustrating total protein input). (B) Lysates from WT MEFs expressing His-Tagged TSPO were used in affinity isolation reactions. Isolated TSPO and VDAC1 were detected with anti-TSPO and anti-VDAC1 antibodies by protein gel blotting. TSPO with the CRAC deletion is still able to interact with VDAC1. Five% of the lysate used for the affinity isolation reaction was loaded for the input and probed with anti-VDAC1 (illustrating VDAC input) and anti-GAPDH (illustrating total protein input). ROS generation was calculated by recording the rate of uptake of the O₂⁻-sensitive dye dihydroethidium (DHE). (C) Depicts representative traces collected in MEFs while (D) summarizes mean uptake rate (P < 0.05; n > 10 cells). (E) Representative images of TSPO-modulated VDAC1^−/- MEFs before and after treatment with FCCP (20 μM) for 4 h. A magnification of the merged images is shown in areas demarcated by the white box. (F) Quantification of the degree of mtRFP:GFP-LC3 colocalization in VDAC1^−/− MEFs (n> 15 cells; P < 0.05).