PBS was needed to be pre-cooled before experiment. After washed with PBS and 1 x RIPA lysis buffer was added to cells (Cell Signaling Technology, Danvers, MA, USA) with 1:100 phosphatase inhibitor and 1:100 protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). 10% SDS-PAGE was used to separate protein and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). At room temperature, membranes were blocked with 5% BSA or 5% milk for 1 h which was then dissolved in TBST buffer containing 0.1% Tween-20. Later we incubated with corresponding primary antibodies overnight at 4 °C and the appropriate secondary antibodies. After washing four times with TBST, protein detection was performed by chemiluminescence (Pierce, Rockford, IL, USA). Antibodies used in this research included anti-MMP2 (D8N9Y), anti-N-cadherin, anti-Vimentin (RV202, BD Biosciences, USA), anti-Snail (C15D3, Cell Signaling Technology, Danvers, MA, USA), and anti-mouse or rabbit secondary antibodies (Cell Signaling Technology).
Anti vimentin rv202
Manufactured by BD
Sourced in United States
Anti-Vimentin (RV202) is a monoclonal antibody that binds to the vimentin protein. Vimentin is a type III intermediate filament protein found in various cell types. The primary function of this antibody is to detect and quantify the presence of vimentin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti n cadherin, by BD (3 mentions)
Anti mouse or anti rabbit secondary antibody, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Anti snail c15d3, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti nrp1 a 12, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin hecd 1, by Abcam (1 mentions)
Anti β actin 13e5, by Cell Signaling Technology (1 mentions)
Anti mad2, by Fortis Life Sciences (1 mentions)
Srx 101a, by Konica Minolta (1 mentions)
Anti p190, by BD (1 mentions)
Anti p ddr1 tyr513, by OriGene (1 mentions)
Anti erm, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
4 protocols using anti vimentin rv202
1
Western Blot Analysis of EMT Markers
2
Immunoblotting of Angiogenesis Markers
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All procedures were performed as we previously reported22 (link). The antibodies used in our current study were anti-NRP1 (A-12) (Santa Cruz Biotechnology, CA, USA), anti-HIF-1α (D1S7W), anti-VE-cadherin (D87F2), anti-MMP2 (D8N9Y) (Cell Signaling Technology, Danvers, MA, USA), and anti-Vimentin (RV202) (BD Biosciences, Oxford, UK). Anti-β-actin and anti-mouse or anti-rabbit secondary antibodies from Cell Signaling Technology were used.
3
Western Blot Analysis of Signaling Proteins
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Western blotting analysis was performed as previously described.13 The antibodies used in the analysis were anti‐NRP1 (A‐12), anti‐phospho (p)EGFR (Tyr1068) (1H12), anti‐EGFR (A‐10) (all from Santa Cruz, Santa Cruz, CA), anti‐pAKT (Ser473) (D9E), anti‐AKT, anti‐pFAK (Tyr397) (D20B1), anti‐FAK (D2R2E), anti‐Cyclin D1 (92G2), anti‐MMP2 (D8N9Y), anti‐MMP9 (603H), anti‐Snail (C15D) (all from Cell Signaling Technology, Danvers, MA), anti‐N‐cadherin, anti‐Vimentin (RV202) (both from BD Biosciences, San Jose, CA), anti‐β‐actin and anti‐mouse or anti‐rabbit secondary antibodies (all from Cell Signaling Technology). According to the protein loading marker, we cut the whole membranes into small pieces and incubated them with corresponding specific primary antibody overnight at 4°C. On day 2, after washing four times with 1 × TBST (Tris‐buffered saline–Tween 20), we continued to incubate the membrane pieces with the appropriate secondary antibodies. Image J was used to quantify the band density of the immunoreactive proteins. After we opening our immunoblot images in image J, we transformed the image type to 8‐bit to be recognized and then subtracted the background. Finally, we set the associated measurements and selected our target band to obtain integrated density value for further analysis in Excel.
4
Western Blotting Protocol for Protein Analysis
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Cells were collected and resuspended in RIPA buffer (Thermo Fisher Scientific) with added protease inhibitors (Roche). Protein concentration was quantified using the Bio-Rad DC protein assay (15 µg loaded per well). Protein samples were resuspended in Laemmli buffer and separated on SDS-PAGE and transferred onto PVDF membranes. Antibodies used included anti–β-actin 13E5 (1:5,000; Cell Signaling Technology), anti–E-cadherin HECD-1 (1:200; Abcam), anti–DDR1 C-20 (1:200; Santa Cruz Biotechnology), anti-KIFC1 (HSET; 1:500; Bethyl Laboratories), anti-Mad2 (1:500; Bethyl Laboratories), anti-RhoE (1:100; Sigma-Aldrich), anti-p190 (1:250; BD Biosciences), anti–STARD8 E-2 (DLC3; 1:100; Santa Cruz Biotechnology), anti–N-cadherin (1:500; BD Biosciences), anti–Vimentin RV202 (1:500; BD Biosciences), anti-ERM (1:500; Cell Signaling Technology), anti–pMLC T18/S19 (1:500; Cell Signaling Technology), and anti–pDDR1 Tyr513 (1:100; Origene). Western blots were developed using a SRX-101A Konica Minolta and scanned.
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