Anti p srctyr416

Whole-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride (PVDF; Millipore, Billerica, MA, USA) membrane using a semidry transfer system (Bio-Rad). The membranes were probed with specific antibodies as indicated and then incubated with horseradish peroxidase-conjugated antibody against mouse or rabbit immunoglobulin (Cell Signaling Technology, Danvers, MA, USA), followed by the detection with enhanced chemiluminescence Western blotting detection reagents (GE Healthcare, Little Chalfont, UK). An ImageQuant LAS4000 mini system (GE Healthcare) was used to detect chemiluminescence. The following antibodies were used for immunological analysis in this study: anti-p-Src^Tyr416 (Cell Signaling Technology, #2101), anti-Src (Sigma, SAB4300433), anti-Akt (Cell Signaling Technology, #4691), anti-p-Akt^Ser473 (Cell Signaling Technology, #4060), anti-p-Akt^Thr308 (Cell Signaling Technology, #13038), anti-PTPN1 (BD Transduction Laboratories, #610139), anti-p-EGFR^Tyr1068 (Cell Signaling Technology, #3777), anti-EGFR (Cell Signaling Technology, #4267), and anti-β-actin (Sigma-Aldrich, A5316) antibodies.

Protein lysates were prepared using RIPA buffer supplanted with a protease inhibitor cocktail. The concentration of protein was determined by the DC Protein Assay Kit. The equal amounts of protein samples were separated on SDS polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. This membrane was blocked with a blocking solution, TBST, supplemented with 5% Difco Skim Milk (BD bioscience) for 1 h, the membrane was then incubated with PAIP1-specific antibody (Santa Cruz), anti-GAPDH antibody (Abcam), anti-α-Tubulin (Santa Cruz), anti-SRC (Cell Signaling Technology), anti-pSRC (Tyr416) (Cell Signaling Technology), or anti-MMP9 (Cell Signaling Technology) in TBST. Following incubation with HRP-conjugated secondary antibodies, proteins were finally detected using the SuperSignal West Pico Chemiluminescent Substrate and captured using ImageQuant LAS 500 (GE Healthcare Life Sciences).

Baicalein (purity > 95%, HPLC) was purchased from Meilun Bio (Dalian, China) and prepared with 0.5% CMC-Na solution. Matrigel was purchased from BD Biocoat (New Jersey, USA). Sodium pentobarbital was from Merck Drugs & Biotechnology (New Jersey, USA). Id1 rabbit monoclonal antibody was from BioChek (San Francisco, USA). Antibodies including anti-E-Cadherin, anti-N-cadherin, anti-vimentin, anti-β-actin, anti-Src, and anti-p-Src(Tyr416) were purchased from Cell Signaling Technology (Massachusetts, USA) and VEGF-A from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit-IgG were from Pierce Biotechnology (Rockford, IL).