Western blots were performed using standard protocols. Cell lysates were prepared using RIPA buffer. Protein samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk. The membrane was then incubated with anti-E-Cadherin antibody (1:1000, bs-10009R, Bioss, Beijing, China), anti-Vimentin antibody (1:1000, bs-0756R, Bioss, Beijing, China), anti-TRAF6 antibody (1:1000, bs-1184R, Bioss, Beijing, China), anti-N-Cadherin antibody (1:1000, AF5237, Beyotime, Shanghai, China), anti-RGS2 antibody (1:1000, sc-100761, Santa Cruz), anti-Flag antibody (1:1000, M185-3, MBL, Beijing, China) or anti-αTubulin antibody (1:5000, AF5012, Beyotime, Shanghai, China) at 4 °C overnight, followed by incubation with HRP-anti-rabbit or HRP-anti-mouse secondary antibody at room temperature for 1 h. Signals were detected using an ECL Kit (P0018AS, Beyotime, Shanghai, China) according to the manufacturer’s protocol.
Bs 0756r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-0756R is a lab equipment product offered by Bioss Antibodies. It is designed for specific laboratory applications. The core function of this product is to provide a reliable and precise tool for researchers and scientists in their laboratory work. However, a detailed description of the Bs-0756R's features and intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sc 100761, by Santa Cruz Biotechnology (1 mentions)
Bs 10009r, by Bioss Antibodies (1 mentions)
Af5012, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab22759, by Abcam (1 mentions)
Bs 0279r, by Bioss Antibodies (1 mentions)
Kgp2100, by Keygen Biotech (1 mentions)
Ab76291, by Abcam (1 mentions)
Ab134155, by Abcam (1 mentions)
Bs 4963r, by Bioss Antibodies (1 mentions)
Streptavidin conjugation kit, by Abcam (1 mentions)
Ab259990, by Abcam (1 mentions)
Ab56918, by Abcam (1 mentions)
Td12217s, by Abmart (1 mentions)
Ps18323s, by Abmart (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Tiangen Biotech (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Gapdh, by Bioss Antibodies (1 mentions)
7 protocols using bs 0756r
1
Western Blot Analysis of EMT Markers
2
Protein Extraction and Western Blot Analysis
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Protein was extracted by Lysis buffer (KGP2100, KeyGEN BioTECH) and the concentration of protein was measured by bicinchoninic acid protein assay kit (KGSK3051, KeyGEN BioTECH). Fifty micrograms of protein was resuspended in sample loading buffer, boiled for 5 min, and electrophoresed on polyacrylamide gel. The proteins were transferred electrophoretically to a nitrocellu-lose membrane, blocked with PBS-Tween (0.05%) in 5% low-fat dry milk solution at room temperature for 2 h, and incubated with specific antibodies FASN (1:5000, 273kDa, ab22759, abcam), L-FABP (1:1000, 15kDa; ab7807, abcam), VEGF ( 1:500, 45kDa; bs-0279R, Bioss), VEGFR-2 ( 1:500, 151kDa; bs-0565R, Bioss), MEK5 (1:500, 49kDa; bs-4124R, Bioss), E-cadherin (1:500, 80kDa; bs-1016R, Bioss), or vimentin (1:500, 51 kDa; bs-0756R, Bioss) overnight at 4°C. The horseradish peroxidase-labeled goat-anti-rabbit (ZB 2301) or goat-anti-mouse (ZSGB-BIO) secondary antibodies were incubated at room temperature for 2 h. ECL (KGP1121, KeyGEN BioTECH) was used for chemiluminescence color reaction.
3
Antibody Modification and DNA Conjugation
4
Immunohistochemical Analysis of Key Proteins
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After paraffin embedding, samples were prepared in 4-μm slices, followed by dehydration and dewaxing overnight in a drying oven at 65 °C and then completely dewaxed in xylene and gradient alcohol solutions. After high-pressure antigen repair with EDTA alkaline or sodium citrate repair solution, the sections were subjected to 12-min incubation with 30% hydrogen peroxide, and another 30-min incubation using goat serum. Then, antibodies against ROD1 (1:500, # ab56918, Abcam, UK), YTHDC1 (1:500, # ab259990, Abcam, UK), OIP5 (1:300, # TD12217S, Abmart, China), GPD1L (1:200, # PS18323S, Abmart, China), N-cadherin (1:400, # bs1172R, Bioss, China), E-cadherin (1:500, # ab40772, Abcam, UK), and vimentin (1:400, # bs0756R, Bioss, China) were supplemented for overnight incubation under 4 °C. The next day, we introduced the secondary antibody for 1-h incubation, DAB color developing solution was supplemented, and hematoxylin was used to restain the nucleus.
5
Western Blot Analysis of Protein Expression
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Total protein was extracted with radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO, USA) and quantified with bicinchoninic acid protein assay kit (Tiangen, Beijing, China). Following separation by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) electrophoresis, the proteins were blotted to polyvinylidene difluoride membranes (Sigma-Aldrich). After that, the membranes were blocked for 1 h with 5% non-fat milk and cultivated overnight with primary antibodies against APPBP2 (bs-11639R; Bioss, Beijing, China), Vimentin (bs-0756R; Bioss), E-cadherin (bs-1519R; Bioss), N-cadherin (bs-1172R; Bioss), and GAPDH (bs-2188R; Bioss) at 4°C. Thereafter, the membranes were kept with horseradish peroxidase-conjugated secondary antibody (bs-0294M-HRP; Bioss) for 2 h at indoor temperature. The immunoblots were visualized by using enhanced chemiluminescence reagent (Beyotime, Shanghai, China).
6
Immunofluorescence Staining of Tumor Sections
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Immunofluorescence staining was performed as recommended by the manufacturer. Briefly, tumor sections with a thickness of 8 μm were fixed with cold acetone for 10 min, dried on slide rack for 30 min, rinsed with ttPBS for 3 times, blocked with 10% goat serum for 60 min at room temperature and then incubated with the primary antibody for α-SMA (Abcam ab32575, 1:100) and vimentin (Bioss, bs-0756R) at r.t. for 2 h. The sections were washed at least 3 times, at least 5 minutes each time in ttPBS and further incubated with FITC or Alexa Fluor 647 labeled goat anti rabbit second antibody (BD, 1:100) for 1 h at r.t. in the dark. Slides were rinsed with ttPBS and PBS for at least 3 times and the nuclei were stained with DAPI (Beyotime, C1006) staining buffer for 15 min. Fluorescent images were acquired by a confocal microscope (Nikon A1).
7
Protein Quantification and Western Blot Analysis
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The total protein was extracted by RIPA, and the protein concentration was determined by the BCA method. Protein was separated on 10% SDS-PAGE gel and transferred to PVDF membrane. Then, the membranes were blocked with 5% nonfat at room temperature for 1 h and washed with TBST membrane for 3 times. The membranes were incubated with the primary antibody anti-CDH2 protein antibody (1RV 1000 diluted, A0432 negative), anti-CDH1 protein antibody (1RV 1000 diluted, 3195 CST), anti-Vimentin protein antibody (1RV 1000 diluted, bs-0756R, BIOSS), anti-α-SMA protein antibody (1RV 1000 diluted, ZB019, YTHX), and β-actin (1PV2000 diluted, AP0060, Bioword) overnight at 4°C. On the second day, the mice were rewarded for 30 min and washed with 10 min for 3 times, and the corresponding secondary antibodies (diluted anti-mouse HRP, 1 : 5000, item number 00001-1, ProteinTech) were added and incubated at room temperature for 3 times. Add ECL reaction for 1 min, absorb the excess photoluminescence solution, expose in darkroom, and scan the strip by scanner.
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