Fig. 2.

Knockdown of RPIA increases ATG4B-mediated cleavage of LC3.

A) Schematic overview of the ATG4B-mediated luciferase release assay. Luciferase is secreted from the cells and a chemiluminescence signal in the supernatant can be measured. B) Luciferase release assay 24 h post-transfection with the indicated plasmids in 293ET cells transduced with the indicated reporter constructs. Data represent mean ± SD, n = 3. C) Luciferase activity of supernatant collected 96 h post-transduction with control (pMOWS) and shRPIA in 293ET cells as indicated. Data represent mean ± SD, n = 3. D, E) Luciferase activity of supernatants in transduced MCF7 (E) and primary murine fetal liver cells (F) collected 96 or 24 h hours post-transduction with control (pMOWS) and shRPIA, respectively. Data represent mean ± SD, n = 3. F) 293ET cells were co-transfected with Flag-RPIA-WT and Flag-RPIA^res (resistant to shRNA knockdown) together with the indicated shRNA constructs. Cell lysates were resolved by PAGE and blotted using a Flag-antibody (top panel) and beta-actin as a loading control (bottom panel). These results demonstrate that RPIA^res is resistant to knockdown with shRPIA. G) Luciferase release assay 96 h post-transfection with control (pMOWS), shRPIA and ± shRNA resistant RPIA in 293ET cells expressing Act-LC3-dNGLuc. Data represent mean ± SD, n = 3.