Anti vinculin antibody

Pancreatic cancer cells were seeded in 6-well plates containing sterilized, collagen I (Invitrogen, Grand Island, NY)-coated coverslips (22×22 mm, Corning) at a density of 2×10⁵ cells/well. The cells were prepared with three groups: 1) control (without treatment); 2) scramble (transfected with negative control siRNA); and 3) hENT1 knockdown. The cells were fixed with 4% paraformaldehyde (Affymetrix, Santa Clara, CA) for 30 min at room temperature and then permeabilized with 2% Triton X-100 (Sigma Aldrich, St. Louis, MO) for 10 min. Following blocking with 2% bovine serum albumin (Calbiochem) for 1 hour, the cells were incubated overnight with anti-Vinculin antibody (1∶200, Abcam, Cambridge, MA), anti-E-cadherin (1∶200), anti-N-cadhrein (1∶100), anti-cytoketarin 18 (1∶200), or anti-Lamin A/C (1∶200) at 4°C with gentle shaking. Then cells were washed twice with PBS for 10 min and incubated with Alexa 488 or 594-conjugated secondary antibodies for an hour at room temperature. After washing with PBS, for F-actin staining, cells were incubated with Alexa Fluor 488 Phalloidin (1∶250, Invitrogen, Grand Island, NY) for 30 min at room temperature. Then, the nuclei were stained using Hoechst 33342 (Molecular probes, Life Technologies, Grand Island, NY) for 10 min. The cells were monitored by confocal laser scanning microscope (CLSM, Olympus FluoView FV1000).

For immunofluorescence, the slides were deparaffinized, rehydrated, soaked in 10 mM sodium citrate buffer, and maintained at 95–99 °C for 10 min. After 30 min of cooling, the slides were washed in distilled water three times for 5 min each and then washed in PBS another three times for 5 min each. The slides were blocked with blocking solution for 1 h at room temperature, and then the primary antibody, including an anti-Vimentin antibody (cat# sc-6260, Santa Cruz, Dallas, TX, USA) and an anti-Vinculin antibody (cat# ab129002, Abcam, Cambridge, UK), was added. The slides were incubated overnight at 4 °C. After washing with PBS three times for 5 min each, the secondary antibody was added, followed by incubation for 1 h at room temperature in the dark. The nuclei were stained with 4′,6-Diamidino-2-phenylindole. The immunofluorescence images of all slides were obtained using a confocal laser scanning microscope (LSM 710, Carl Zeiss, DE, Jena, Germany), available at the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute (KBSI), and immunofluorescence intensity was quantified from two random fields of view from twelve biological replicates per group at the 10× magnification using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Cell lysates were prepared in M-PER buffer (Thermo Fisher Scientific, 78501) supplemented with protease inhibitors (Roche). For Western blotting, cell lysates and EVs were resuspended in NuPAGE LDS sample buffer (Novex, NP0008) with NuPAGE sample reducing agent (Novex, NP0009). IP was carried out using the FLAG immunoprecipitation kit (Sigma-Aldrich). Cell lysates were incubated in anti-FLAG agarose affinity gel at 4°C overnight. Immunoprecipitated samples were washed three times with washing buffer, eluted, and subjected to Western blotting. All Western blotting samples were loaded on a 4 to 12% NuPAGE gel or 12% NuPAGE gel and transferred onto 0.22-μm polyvinylidene difluoride membrane (Bio-Rad, 1620177). Primary antibodies include anti-FLAG antibody (Sigma-Aldrich, F1804; 1:2000 dilution), anti-vinculin antibody (Abcam, ab129002; 1:2000 dilution), anti-CD9 antibody (Cell Signaling Technology, 1317S; 1:1000 dilution), anti-Scamp3 antibody (GeneTex, GTX102216; 1:2000 dilution), and anti-ARRDC1 antibody (in-house; 1:3000 dilution). Secondary antibodies include horseradish peroxidase (HRP)–conjugated anti-rabbit (Cell Signaling Technology, 7074S; 1:2000 dilution) and HRP-conjugated anti-mouse (Cell Signaling Technology, 7076S; 1:2000 dilution).

Protein from the oviduct magnum tissue (positive control), chicken leg muscle tissue (negative control), DF-1 (negative control), and cOECs at passage 2 were extracted using RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Scientific, USA). The protein concentration was determined with the Bradford assay (Bio-Rad, Hercules, CA, USA). Protein extracts (15 μg) was electrophoresed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis 4% to 12% gel system (Invitrogen, USA) and transferred to polyvinylidene fluoride (Invitrogen, USA) membrane. The membrane was blocked with 5% skim milk in PBS (Thermo Scientific, USA) for 1 h and then primarily incubated with rabbit polyclonal anti-ovalbumin antibody (1:1,000 [1 mg/mL] dilution, Abcam, UK) and rabbit monoclonal anti-vinculin antibody (1:1,000 [0.054 mg/mL] dilution, Abcam, UK) overnight 4°C. The membrane was washed and then secondarily incubated with mouse anti-rabbit HRP conjugated antibody (1:2,000 [0.4 mg/mL] dilution, Santa Cruz, Dallas, TX, USA) for 30 min at room temperature. Amersham ECL prime (GE Healthcare, Buckinghamshire, UK) substrate was used to visualize the target bands, and the bands were analyzed using EZ-Capture II (Atto, Tokyo, Japan).

The visualization of the desired focal adhesion (FA) proteins including Vinculin and FAK was carried out by immunofluorescent staining. The cells were seeded on different surfaces at the density of 1 × 10⁵ cells per sample and cultured for 24 h. Then the cells were gently rinsed with phosphate-buffered saline (PBS; HyClone, USA) and fixed with 4% paraformaldehyde. After permeabilized with 0.1% Triton X-100 for 10 min, the cells were blocked with 5% goat serum albumin (Boster, USA) for 30 min. Then the cells were incubated with anti-vinculin antibody (1:100, Abcam) or anti-FAK antibody (1:400, Proteintech, USA) at 4 ℃ overnight. The cells were labeled with Dylight 488, goat anti-rabbit IgG second antibody (1: 200, Abbkine, USA) for 2 h at 37 ℃. Finally, the cells were mounted by Antifade Mounting Medium with DAPI (Beyotime) and imaged using confocal laser scanning microscope (Nikon Instruments Inc.). The fluorescent intensity of the cells was analyzed using the ImageJ software.

Western blotting was employed to detect the expression levels of ER‐α and vinculin in MCF‐7 cells treated with fulvestrant (0 and 10 nM). In brief, the cells were first rinsed with pre‐chilled 1× PBS buffer (pH 7.4) to remove soluble factors, then lysed with radio immunoprecipitation assay lysis (RIPA) buffer containing 0.5 M Tris‐HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP‐40, 10 mM EDTA, and protease inhibitor mixture. The total protein mixture was separated through SDS‐PAGE and transferred to polyvinylidene difluoride membranes using a Bio‐Rad semi‐dry electrophoretic cell. After the membranes were incubated with anti‐ERα antibody (dilution 1:750 in 1× PBS; Abcam, USA) and anti‐vinculin antibody (dilution 1:400 in 1× PBS; Abcam, USA), the target antibodies were labeled with a horseradish peroxidase (HRP)‐conjugated IgG antibody. Finally, the enhanced chemiluminescence (Pierce) was utilized for immunoreactive protein visualization.

In all, 2 × 10⁶ cells were lysed using either RIPA buffer or CellLytic M (Sigma) with a HALT protease inhibitor cocktail (Thermofisher). Protein lysates were separated on SDS gel and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h with PBS-T containing 5% skim milk and were incubated at 4 °C overnight with a primary antibody. Membranes were washed with PBS-Tween (0.05%) and incubated with a secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories) for 2 hr at room temperature. The membrane was rewashed with PBS-T. Proteins were detected using enhanced chemiluminescence (ClarityTM Western ECL Blotting Substrates Bio-rad), and signals were detected using a gel documentation system (Bio-rad). For ERG expression and cellular sub-localization: 293T cells were transiently transfected with ERG expression vectors using the calcium phosphate Profection Mammalian Transfection kit (Promega). 48 hours following transfection, cells were collected, and cytoplasmatic or nuclear proteins were extracted using NucBuster Protein Extraction Kit (Novagen) according to the manufacturer’s instructions.
For ERG immunoblot we used anti-human ERG antibody by Santa Cruz (C-17, sc-354, 1:1000). For HA immunoblot we used anti-HA monoclonal antibody (Sigma, H3663). For Vinculin immunoblot we used anti-Vinculin antibody manufactured by Abcam.