Figure 2. PPP1R15 selectively associates with monomeric G-actin in cells.
(A) Immunoblot (upper panel) and Coomassie-stained gel (lower panel) of affinity-purified GFP-tagged PPP1R15A and purified actin. Samples were incubated and centrifuged to pellet F-actin (lane 1), leaving G-actin in the supernatant (lane 2); pellet P, supernatant S. (B) Immunoblot for GFP and actin of GFP-affinity purified proteins (upper two panels) from HEK293T cells expressing GFP-tagged PPP1R15A (hR15A-GFP) treated with 2 µM of each indicated compound. Immunoblot for actin of 2% of input. (C) Fluorescence microscopy image of NIH-3T3 cell F-actin arrangement. NIH-3T3 cells were left untreated (control), cultured in serum-free medium for 24 hr (serum starved), cultured in serum-free medium for 18 hr, followed by addition of medium containing 10% vol/vol FBS for 6 hr (serum refed), then fixed and stained with Alexa-Fluor 568 phalloidin and imaged by confocal microscopy. (D) Immunoblot for GFP and actin of NIH-3T3 lysates from cells treated as in ‘C’ then subjected to GFP affinity purification (upper two panels). Immunoblot for actin of 2% of input.