Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot