Anti vimentin

Western blot analysis was conducted as previously described [24 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, and anti-c-Met (Epitomics, Burlingame, CA, USA), anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin, anti-c-myc, anti-EGFR, anti-p-EGFR (Tyr1068), anti-GSK3β, anti-p-GSK3β (Ser9), anti-AKT (pan), anti-p-AKT (Ser473), anti-stat3, anti-p-stat3 (Tyr705) and anti-CREB1 (Cell Signaling Technology, Beverly, MA).

Immunocytochemistry was done on PFA 4 % fixed cells, and stained with the following antibodies: the primary antibodies were anti-E-Cadherin (BD Biosciences 610181), anti-HNF4a (Abcam ab41898), and anti-Vimentin (Epitomics, 2707-1); the secondary antibodies were Alexa-Fluor 488 and Alexa-Fluor 594, from Molecular Probes. The nuclei were stained with NucRed® Live 647 (Catalog number: R37106, Life Technologies), and preparations were analyzed by confocal microscopy (Leica TSC SP8).

Du-145 prostate cancer cells grown on cover slip were fixed in 4% paraformaldehyde (Invitrogen, Cergy-Pontoise, France) for 15 minutes (min). After washed three times with PBS, unspecific sites were blocked using PBS containing 5% BSA at room temperature for 1 h. Cells grown on cover slip were then incubated with anti-E-cadherin (Cell Signaling Technology, Beverly, MA), anti-N-cadherin (Cell Signaling Technology, Beverly, MA), and anti-Vimentin (Epitomics, Burlingame, USA) at 4°C overnight, and incubated with the secondary antibodies Alexa Fluor (Molecular Probes, Life Technologies, Saint-Aubin, France) in the dark at room temperature for 1 h. DAPI were used for Nuclei staining. All stained cells were examined and photographed with a Leica SP5 confocal fluorescence microscope.

SW480 and HCT116 cells were seeded in 6-well plates until they grew with adherence, the various conditioned media was replaced as described previously and incubated for 24 hours, and cell extracts were prepared in ice-cold lysis buffer containing protease inhibitor. The cell proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride (PVEF) membranes (Millipore). Membranes were further incubated sequentially with specific antibodies including anti-E-cadherin (1:1000, Pro780, Cell Signaling Technology), anti-N-cadherin (1:1000, EPR1791–4, Epitomics), anti-Vimentin (1:1000, EPR3776, Epitomics), anti-MIF (1:1000, Santa Cruz Biotechnology), anti-p-Cofilin (1:1000, 77G2, Cell Signaling Technology), and anti-F-actin (5 μg/ml, 4E3.adl, Abcam). After primary antibodies were incubated, the blots were subsequently incubated with appropriate secondary antibodies. Protein bands were visualized with ECL reagent (Thermo Scientific Inc.) and a Bio-Rad image acquisition system (Bio-Rad Laboratories). The protein bands was quantified using densitometric scanning software, and relative protein abundance was determined by normalization with tubulin or GAPDH.

A total of 50 mg protein was separated by denaturing 8–15% SDS–polyacrylamide gel electrophoresis. Western analysis was conducted as previously described [12] (link). The films were analyzed by densitometry with a VersaDoc Imaging System; Model 3000 (BioRad) using Quantity One software. Analysis of variance with Bonferroni correction for multiple tests was used to determine significance. The primary antibodies used were anti-Ncadherin (1∶5000), anti-Ecadherin (1∶2000), anti-fibronectin (1∶1000), anti-vimentin (1∶1000) and anti-actin (1∶2000) as an endogenous control, all from Epitomics Biotechnology (Epitomics).

Cells were cultured at a density of 1.5 × 10⁵ cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.

Gastric cancer cells grown on cover slip were fixed in 4% paraformaldehyde (Invitrogen, Cergy-Pontoise, France) for 15 minutes (min). After washed three times with PBS, unspecific sites were blocked using PBS containing 5% BSA at room temperature for 1 h. Cells grown on cover slip were then incubated with anti-N-cadherin (Cell Signaling Technology, Beverly, MA), anti-Fibronectin and anti-Vimentin (Epitomics, Burlingame, USA) at 4°C overnight, and incubated with the secondary antibodies Alexa Fluor (Molecular Probes, Life Technologies, Saint-Aubin, France) in the dark at room temperature for 1 h. DAPI were used for Nuclei staining. All stained cells were examined and photographed with a Leica SP5 confocal fluorescence microscope.