Journal: PLoS ONE
Article Title: Resistance to Inhibitors of Cholinesterase (Ric)-8A and Gα i Contribute to Cytokinesis Abscission by Controlling Vacuolar Protein-Sorting (Vps)34 Activity
doi: 10.1371/journal.pone.0086680
Figure Lengend Snippet: ( A ) Vps34, GFP and actin immunoblots of cell lysates immunoprecipitate from HeLa cells transiently transfected with vectors expressing YFP, Gα i3 wt-YFP, or Gα i3 QL-YFP. Experiments were repeated twice with similar results. ( B ) 3D reconstruction of confocal images of HeLa cells expressing GFP-Ric-8A immunostained for endogenous Vps34. Scale bar is 5 µm. The right panels show an electronic magnification of midbody area (white square). ( C–D ) Co-immunoprecipitation of endogenous Vps34 and endogenous Ric-8A in HeLa cells. The experiment was repeated 3 times with similar results. ( E ) Asynchronous HeLa cells lysates treated with PTX (200 ng/mL, 3 h) or with its vehicle were immunoprecipitated using anti-Vps34 antibody and immunoblotted for Ric-8A or Vps34. ( F ) Quantification of PtdIns(3)P production after purification of endogenous Vps34 kinase in asynchronous or G2/M enriched HeLa cells treated with PTX (200 ng/mL, 3 h) or siRNA control versus siRNA Ric-8A. Quantification was performed from 4 independent experiments (*, p<0.05).
Article Snippet: Primary antibodies used for western blotting were the following: anti Actin-HRP conjugated (Sigma, 1∶20,000), anti GFP (Cell Signaling, 1∶1,000), anti cyclinB1 (Cell Signaling, 1∶1,000), anti phospho-p190-progesterone receptor (Invitrogen, 0.5 µg for IP), anti-ubiquitin K63 linked-HRP conjugated (Enzo, 1∶1,000); anti total ubiquitin-HRP conjugated (Santa Cruz, P4D1, 1∶1,000); anti-Gα i3 rabbit polyclonal (Santa Cruz, 1∶500), anti-Gβ 1 rabbit polyclonal (Santa Cruz, 1∶50), anti-Gγ5 rabbit polyclonal (Santa Cruz, S14, 1∶100), anti-Ric-8A rabbit polyclonal (kind gift from Dr. G. Tall, University of Rochester, 1∶20,000 or Abcam, 1∶1,000 for WB or 0.5 µg for IP), anti-phospho Histone H3-Serine 10 (Cell signaling, 1∶1,000), anti-phospho serine (Cell signaling, 1∶300), anti-Vps34 (Echelon, 1∶1,000) and anti-Vps15 (Bethyl, 1 µg for IP and Abcam, 1∶1000 for WB).
Techniques: Western Blot, Transfection, Expressing, Immunoprecipitation, Purification