Anti phospho paxillin tyr118

Due to unique cellular responses observed in HFLS-RA cells (compared to HFLS-OA or HFLS control cells), cell signaling pathways were examined only in HFLS-RA cells. Several signal transduction pathways involved in the transformation of HFLS-RA cells to an aggressive phenotype were examined (16 (link), 50 (link), 59 (link)). Briefly, HFLS cells were incubated with antigens for 15-min, 30-min, 1-h and 4-h, cellular lysates were prepared as above, and the following primary antibodies (all polyclonal rabbit IgGs) were utilized to probe the samples; anti-β-actin antibody (endogenous control) (Novus Biologicals), anti-SAPK/JNK (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-Erk1/2 (p44/42 MAPK) (#9102), anti-phospho-Erk1/2 (p44/42 MAPK; Thr202/Tyr204) (#9101), anti-Akt (#9272), anti-phospho-Akt (Ser473) (#9271), anti-p38 MAPK (#9212), anti-phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-Paxillin (#2542), anti-phospho-Paxillin (Tyr118) (#2541), anti-FAK (#3285), anti-phospho-FAK (Tyr397) (#3283) (Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch) was used as a secondary antibody. Respective band intensities were measured and reported as outlined above. The maximum and minimum activation of signaling pathways for HFLS lysates was determined based on positive (treatment with LPS) and negative (treatment with media) controls.

The anti-FNDC4, anti-MYOG and anti-desmin were from Bioss Biotechnology. The anti-GAPDH, anti-Phospho-paxillin (Tyr118), anti-paxillin, anti-Phospho-FAK (Tyr925), anti-FAK, anti-Vinculin and anti-ITGβ1 were purchased from Cell Signaling. The anti-6× his and anti-IgG were from Santa Cruze Biotechnology. The secondary antibodies were HRP-labeled and FITC-labeled were from Beijing Biosynthesis Biotechnology Co., Ltd. PF562271 was from Selleck and used concentration for 10 nM/ml to treat cells.

Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins by collagenase treatment as described previously [36 ]. Cells from passages 2–7 were subsequently used in experiments. HUVECs were grown in M199 media (Welgene, Daegu, Korea) supplemented with 20% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, 3 ng/ml of basic fibroblast growth factor (Millipore, Billerica, MA, USA), and 5 units/ml heparin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C under a humidified 95% and 5% (v/v) mixture of air and CO₂. LS174T human colorectal carcinoma cells and dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary DG44 (CHO-DG44) cells were grown in Dulbecco's modified eagle media (DMEM) or minimum essential media (MEM)-α (Gibco, Grand Island, NY, USA), respectively, supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS or 10% dialyzed FBS (for CHO-DG44 cell). VEGF₁₆₅ and mouse endostatin (mEndo) were purchased from R&D Systems (Minneapolis, MN, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). Anti-human IgG and anti-mouse endostatin antibodies were purchased from Sigma. Anti-CD31, phospho-VEGF receptor 2 (Tyr¹¹⁷⁵), pSrcTyr⁴¹⁶, pERK1/2, pFAKTyr³⁹⁷, and anti-phospho-paxillin Tyr¹¹⁸ were purchased from Cell Signaling (Beverly, MA, USA).