KYSE-150 and KYSE-150R cells were collected and lysed using RIPA Lysis and Extraction Buffer (Thermo Scientific), and protein concentration was measured by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skim milk at room temperature for 2 h, then incubated with primary antibodies against E-cadherin (1:10000; ab40772, Abcam), vimentin (1:2000; ab92547, Abcam), snail (1:200; ab82846, Abcam), β-catenin (1:5000; ab227499, Abcam), Wnt3 (1:1000; ab32249, Abcam), β-actin (1:5000; ab227387, Abcam), histone H3 (1:1000; MA5-15150, Abcam) overnight at 4°C. The membrane was subsequently incubated with HRP-conjugated secondary antibodies (1:5000) at room temperature for 1.5 h. The proteins were visualized using enhanced chemiluminescence (Thermo Scientific), and the band intensity was determined by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
Ab32249
Manufactured by Abcam
Sourced in United States
Ab32249 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab227499, by Abcam (1 mentions)
Ab227387, by Abcam (1 mentions)
Ab82846, by Abcam (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab92547, by Abcam (1 mentions)
Goat anti actin, by Santa Cruz Biotechnology (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Ab94915, by Santa Cruz Biotechnology (1 mentions)
Sc 2033, by Santa Cruz Biotechnology (1 mentions)
Sc 1616, by Santa Cruz Biotechnology (1 mentions)
Ab32572, by Abcam (1 mentions)
P0013, by Beyotime (1 mentions)
Ab63856, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
Ab16051, by R&D Systems (1 mentions)
5 protocols using ab32249
1
Western Blot Analysis of EMT Markers
2
Western Blotting Protocol for Protein Expression Analysis
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Western blotting was performed using established methodology [13 (link)]. Briefly, cells were washed with PBS and lysed in RIPA buffer. Proteins (20–30 μg) were separated by 10–12% SDS/PAGE and then transferred on an immunoblot PVDF membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% nonfat milk, the membrane was incubated with primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Technology, sc-2033 for anti-goat, and sc-2004 for anti-rabbit, 1:3000) and then developed using ECL solution (Pierce). Primary antibodies used were rabbit anti-β-catenin (Abcam, ab32572, 1:1000), rabbit anti-fibronectin (Santa Cruz Technology, sc-9068, 1:1000), rabbit anti-Wnt3 (Abcam, ab32249, 1:1000), rabbit anti-Wnt7B (Abcam, ab94915, 1:1000), and goat anti-actin (Santa Cruz Technology, sc-1616, 1:3000). For protein expression quantitation, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image/ ).
3
BMSC Osteogenic Differentiation Pathway
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After BMSCs were treated with the different drugs, cell lysis buffer for WB and IP (P0013, Beyotime Biotechnology, China) was used to collect the total protein from the cells. The expression of relative proteins was detected by WB according to the protocol we described[1]. The primary protein antibodies were as follows: anti-RUNX2 (1:1000, 12556, CST), anti-Osteopontin (1:1000, ab63856, Abcam), anti-collagen I (1:1000, ab260043, Abcam), anti-β-actin (1:2000, 3700, CST), anti-β-catenin (1:1000, 8480, CST), and anti-Wnt3 (1:1000, ab32249, Abcam). HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies were used (7074, 7076, CST, USA).
4
Protein Expression Analysis in Trophoblast Cells
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The total proteins of B6Tert, HTR8/SVneo, and JEG-3 cells were collected and cracked by high RIPA buffer (Solarbio, Beijing, China). The concentration of proteins was detected using a BCA kit (Yeasen, Shanghai, China). Then, the SDS-PAGE was used to separate the proteins, and the proteins were transferred to a nitrocellulose (NC) membrane (Haoran, Shanghai, China). The NC membrane was blocked by 5% non-fat milk at room temperature for 1.5 h, and then incubated with the primary antibodies (anti-BRIT1, Abcam, ab121277, dilution: 1: 900; anti-matrix metalloproteinase-2 (MMP-2), R&D, IC903G, dilution: 1: 700; anti-MMP-9, Abcam, ab38898, dilution: 1: 1000; anti-tissue inhibitor of metalloproteinases-1 (TIMP-1), R&D, IC970G, dilution: 1: 700; anti-TIMP-2, R&D, MAB971, dilution: 1: 500; anti-Wnt2, Abcam, ab27794, dilution: 1: 600; anti-Wnt3, Abcam, ab32249, dilution: 1: 600; anti-β-catenin, Abcam, ab16051, dilution: 1: 600; anti-GAPDH, R&D, MAB5718, dilution: 1: 800) at 4°C overnight. Subsequently, the NC membrane was incubated with the secondary antibodies at room temperature for 1.5 h (goat anti-mouse IgG, Abcam, ab6785, 1: 8000; donkey anti-rabbit IgG, R&D, NL004, 1: 5000; mouse anti-rabbit IgG, Invitrogen, BA1034, 1: 7000). Chemiluminescence detection was carried out using ECL reagent (Huiying, Shanghai, China).
5
Whole Cell and Nuclear Protein Extraction
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Whole cell lysates and nuclear protein extracts were prepared as described previously.23 (link) The primary antibodies used were Epithelial-Mesenchymal transition (EMT) Antibody Sampler Kit (#9782, Cell Signaling Technology, Beverly, MA, USA), antibodies against WNT3 (ab32249) and WNT5B (ab94914) from Abcam, and antibodies against PCNA (PC10), β-actin (C11), Lamin B (C-20) and α-tubulin (D-10) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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