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. 2011 Dec 1;7(12):1434–1447. doi: 10.4161/auto.7.12.17793

Figure 4.

Figure 4.

Novel ATG12-ATG3 conjugate and ATG3 dependent LC3 lipidation. (A) Immunoprecipitation and immunoblotting analysis of the new ATG12 conjugate. NIH-3T3 and NIH-3T3 cells stably expressing FH-ATG12 were left untreated or infected with vaccinia virus at a MOI of 3 and harvested 8 h post infection. Lysates were immunoprecipitated with α-FLAG. The protein complexes were separated using SDS-PAGE and immunoblotted with anti-ATG12 or anti-ATG3 antibodies, as indicated. (B) Immunoblotting assay of wild-type, atg5−/−, and atg7−/− MEF cells with an anti-ATG3 antibody. Indicated cell lines were either untreated, amino acid and serum deprived for 2 h with Hank’s media or infected with vaccinia virus at a MOI of 3 and harvested 24 h after infection. Actin levels serve as a loading control. (C) Immunoblotting of wild-type, atg5−/− and atg3−/− MEF cells with a LC3 antibody. Indicated cell lines were either untreated, amino acid and serum deprived for 2 h with Hank’s media or infected with vaccinia virus at a MOI of 3 and harvested 24 h post infection. Lysates were probed with anti-LC3 and anti-actin antibodies. The results are representative of three independent experiments. (D) Quantification of LC3-II to actin ratio in (C). The data are the means ± SD from three independent experiments.