Figure 4. LC3-II lipidation assays with SCOC mutants.

Overexpression of SCOC mutants does not increase LC3-II levels. LC3-II lipidation was monitored by immunoblotting of lysates obtained from PC12 cells expressing either EmGFP alone or EmGFP-SCOC variants. Transfected cells were either maintained in nutrient conditions (N) or starved for 2 hours (S) to induce autophagy. In addition, inhibition of LC3 degradation after autophagic induction was effected by the addition of 100 nM bafilomycin. A smaller fragment recognized by the GFP antibody was detected during starvation in all EmGFP-SCOC constructs, apart from the E93V/K97L mutant. However, basal autophagy appears unaffected by this degradation product because increased levels of LC3-II were observed in cells treated with bafilomycin where LC3-II turnover is blocked.