Fig. 3. LC3-II is a substrate of HsAtg4B. (A) LC3-II is cleaved by HsAtg4B in vitro. Lysate from amino-acid-starved ES cells was mixed with lysate from yeast cells expressing FLAG-tagged HsAtg4A, HsAtg4B, HsAtg4B^C74S, or vector alone, and incubated for 1 hour at 37°C. After the addition of SDS-PAGE sample buffer to stop the reaction, the products were resolved by SDS-PAGE and subjected to immunoblot using antibody against LC3. The data were shown from the two independent experiments (Exp. 1 and 2). (B) The amount of LC3-II is overestimated by immunoblotting. Total cell lysates (T) were prepared from HeLa cells expressing His-Myc-LC3 after starvation for 1 hour and subjected to immunoprecipitation using anti-Myc antibody (IP). The immunoprecipitates were assayed by immunoblotting (lanes 1-4) or CBB staining (lanes 5 and 6). As controls, mock-transfected cells were used.