Journal: Molecular Biology of the Cell
Article Title: The Roles of Two Distinct Regions of PINCH-1 in the Regulation of Cell Attachment and Spreading
Figure Lengend Snippet: Association of Rsu-1 and PINCH-1. (A) 293T and HA-PINCH-1-expressing 293T cells were lysed and affinity precipitated using anti-HA affinity matrix. The precipitates were separated by SDS-PAGE and silver stained. Each band was excised and identified by LC-MS/MS. (B) 293T cells were transfected with HA-tagged FL, ΔLIM1, ΔC, and LIM1 and immunoprecipitated with anti-HA antibody. Immunoprecipitates were probed for Rsu-1 and HA. (C) 293T cells were transfected with myc-tagged Rsu-1, and cell lysates were affinity precipitated with GST and fusion proteins of GST and the indicated fragments. Precipitates were subjected to immunoblotting with anti-myc antibody. The lower panel shows Coomassie blue staining of GST fusion proteins. LIM5: aa251–304, LIM5+C: aa251–325, C: aa305–325. (D) Schematic representation of Rsu-1 deletion mutants that were used in the experiments: FL, aa1–277; LRR, aa1–202; C, aa203–277. (E) 293T cells were transfected with myc-tagged full-length or mutant Rsu-1, and cell lysates were affinity precipitated with a fusion protein of GST and LIM5+C. Precipitates were subjected to immunoblotting with anti-myc antibody. (F) FL cells were seeded onto fibronectin-coated coverslips and fixed 20 min, 60 min, or 8 h later to visualize the localization of GFP-PINCH-1 and Rsu-1 during cell spreading (scale bar = 20 μm): 20 min, attached and round; 60 min, initial spread; 8 h, stable spread. (G) shCtrl and shPINCH cells were immunostained with anti-vinculin and anti–Rsu-1 antibodies (scale bar = 20 μm).
Article Snippet: Equal protein quantities were separated on SDS-polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to PVDF membrane (Millipore, Billerica, MA).
Techniques: Expressing, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transfection, Immunoprecipitation, Mutagenesis