Figure 5. GAA expression and autophagy induction in transiently expressed HEK293 cells. (A) Western blot analysis of GAA protein expression in cells and culture medium that were harvested at 48 hour after transfection. Different molecular forms of GAA protein, i.e., 110kD precursor, 95 kD partially processed intermediate and 76 kD mature GAA were separated by SDS-PAGE and visualized by immunoblotting. Top panel: cell lysates; bottom panel: culture media. (B) Representative immunofluorescent images of the LC3-positive autophagic puncta in HEK293 cells with different transfection. Scale bar: 10μm. (C) Quantification of the % of cells contains ≥ 5 LC3 puncta. (D) Western blot analysis of LC3 and p62 in transfected cells.