Vinculin v9131

Lysates were prepared from cell lines, lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 70% Tergitol, 0.1% SDS, 1 mM Mg₂Cl and protease inhibitor cocktail Sigma-Aldrich (St. Louis, MO). Lysates were centrifuged at 12,000 × g for 15 minutes at 4° C. Proteins were solubilized in sample buffer (Life Technologies) and subjected to SDS-PAGE. Proteins were electroblotted onto PVDF membranes and were immunoblotted with the following antibodies: p15INK4b (K-18), p16INK4a (50.1), IFN-γRα (C-20), HER3 (C-17) all from Santa Cruz Biotechnology (Santa Cruz, CA); Vinculin (V9131) from Sigma-Aldrich; EGFR (D38B1), HER2 (29D8), HER3/ErbB3 (1B2), cleaved caspase-3 (Asp175), TNF-R1 (C25C1), phospho-Akt (Ser473), Phospho-Stat1 (Tyr701) (58D6), phospho-Stat1 (Ser727), phospho-Stat3 (Tyr705) (M9C6), phospho-Jak1 (Tyr1022/1023), phospho-Jak2 (Tyr1007/1008) (C80C3), Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) and β-actin (13E5) from Cell Signaling Technologies (Danvers, MA). After washing, membranes were incubated with HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA). Bands were visualized and quantified by using the enhanced chemiluminescence (ECL) western blot detection system and the Image Reader LAS-1000 Lite version 1.0 software (Fuji). Quantification of western blots was performed using ImageJ software (http://rsb.info.nih.gov/ij/).

HUVECs were fixed with 2% PFA after treatment for 24 h with DMSO or KCH-1521, and blocked with 5% normal goat serum (NGS; #16210, Gibco) in 1× PBS + 0.1% Tween 20 (PBST) for 1 h. The cells were incubated for 1 h at room temperature (RT) with the following primary antibodies: talin (T3287), vinculin (V9131, both from Sigma-Aldrich), and paxillin (610051, BD Biosciences, San Jose, CA, USA). After washing, the cells were incubated for 1 h at RT with Alexa Fluor 594-conjugated anti-mouse IgG (A11005) and Alexa Fluor 488-conjugated Phalloidin (A12379, both from Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; D9542, Sigma-Aldrich). Negative controls for immunofluorescence staining were used to evaluate the specificity of primary antibodies and to exclude the possibility of non-specific staining of secondary antibodies by omitting the incubation of primary antibodies [37 (link)]. All immunofluorescent images were obtained using the TE-FM Epi-fluorescence system attached to an inverted microscope (BX61, Olympus, Tokyo, Japan).

To determine relative differences in protein levels, 2×10⁶ cells were harvested at the indicated time points. Protein samples extracted from total cell lysates using Laemmli buffer were subjected to electrophoresis in polyacrylamide SDS gels under reducing conditions, and subsequently transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Protein bands were detected using the ECL system (GE Healthcare, Buckighamshire, UK). Primary antibodies included: RAB7 (Clone RAB7-117); α-Tubulin (clone DM1A) and Vinculin (V9131) from Sigma (St Louis, MO, USA); pan-Ras (Pan-Ras (Ab-3)) from Calbiochem; AKT and p-AKT (Ser473) from Cell Signaling (Danvers, MA, USA); HRP-conjugated secondary antibodies were from GE Healthcare (Little Chalfont, Buckinghamshire, UK); and anti-goat-HRP, from Jackson Immunoresearch (West Grove, PA, USA). α-Tubulin or Vinculin blots were used as loading controls.

After 48 h from Myc-tagged XIAP or Myc-tagged eGFP plasmids transfection HEK293-FT cells were pre-treated with FC2 at a final concentration of 20 µM. A parallel set of cells was left untreated as control. After 30 min, cells were treated with TNF-α (50 ng/ml). After further 30 min, cells were harvested, washed with ice-cold PBS and lysed in E1A lysis buffer (ELB; 150 mM NaCl, 50 mM Hepes, pH 7.5, 5 mM EDTA, 0.5% NP40) supplemented with the mix of protease inhibitors at 4 °C for 30 min. Samples were clarified at 4 °C by centrifugation for 10 min at 13,000 rpm and then quantified with Bradford Assay. 1 mg of total protein extracts were incubated overnight at 4 °C with Protein-A-Sepharose (Sigma-Aldrich) and anti-Myc-Tag antibody #2278 (dilution 1:200, Cell Signaling Technology, Danvers, MA, USA). The Protein-A-Sepharose was previously washed with ELB buffer three times. The bound proteins and 50 µg of each lysate were analyzed by SDS-PAGE and immunoblotting with anti-human primary antibodies recognizing: XIAP #610763 (BD Biosciences, Franklin Lakes, New Jersey, USA), TAB1 #3226, TAK1 #5206 (Cell Signaling Technology, Danvers, MA, USA) and Vinculin #V9131 (Sigma-Aldrich).

All reagents and chemicals were purchased from Sigma-Aldrich unless otherwise specified. Human, adult, normal spleen (NB820-59259) and testis (NB820-59266) lysates and spleen (NBP2-30202), testis (NBP2-75940), skeletal muscle (NBP2-77813) and ovary (NBP2-30190) tissue slides were from Novus Biologicals. Human, adult, normal ovary lysate (HT-406-ZY) was from BioCat. The β-actin (A1978), the vinculin (V9131) and the α-smooth muscle actin (A5228) antibodies were from Sigma-Aldrich, the vWF polyclonal antibody (PA516634), the Alexa Fluor Plus 488 anti-mouse secondary antibody (A32766), the Alexa Fluor 568 anti-rabbit secondary antibody (A10042) and the HRP conjugated anti-mouse secondary antibody (31,432) were from Thermo Fischer Scientific. The mouse IgG1 antibody (554,121) was from BD Biosciences, the calnexin antibody (2679 T) was from Cell Signaling Technology.