phosphorylated src y416 antibody  (Cell Signaling Technology Inc)

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

Journal: Journal of Cellular and Molecular Medicine

Article Title: MG53 inhibits angiogenesis through regulating focal adhesion kinase signalling

doi: 10.1111/jcmm.16777

Figure Lengend Snippet: rhMG53 inhibits FAK‐Src interaction and Src, Akt and ERK1/2 activation. A, HUVECs were stimulated with rhMG53 (10 μg/mL) or vehicle control for 24 h. Cell lysates were prepared and immunoprecipitated with anti‐FAK antibody. Then, captured proteins were analysed by Western blotting with anti‐Src and anti‐FAK antibodies as indicated. Representative images of 3 independent experiments are shown. B and C, HUVECs were exposed to rhMG53 (0, 5, 10 and 20 μg/mL) for 24 h. Phosphorylation of Src Y416 , Akt T308 and ERK1/2, and total Src, Akt and ERK1/2 were analysed by Western blotting and representative images of 3 independent experiments are shown. The densitometric analysis of phosphorylated Src Y416 , Akt and ERE1/2 normalized to total Src, Akt and ERK1/2 was performed. All data shown are mean ± SD for 3 experiments and are expressed as fold changes. * P < .05; ** P < .01; *** P < .001

Article Snippet: Antibodies to phosphorylated FAK Y397 , FAK, phosphorylated Src Y416 , Src, phosphorylated Akt T308 , Akt, phosphorylated ERK1/2 and ERK1/2 were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Control, Immunoprecipitation, Western Blot, Phospho-proteomics