Proteins were separated by SDS‐PAGE and transferred onto nitrocellulose membranes. The membranes were incubated overnight with primary antibodies. The antibodies we used include rabbit antibodies by Abcam (Cambridge, MA, USA): anti‐osterix (polyclonal), anti‐C/EBPa (monoclonal), anti‐β‐catenin (monoclonal), anti‐opsteopontin (monoclonal) and anti‐ALP (monoclonal); rabbit antibodies by Cell Signalling Technology (Danvers, MA, USA): anti‐PPARγ (monoclonal), anti‐LRP6 (monoclonal) and anti‐Phospho‐LRP6 (polyclonal); mouse mAb by MBL (Nagoya, Japan): anti‐Runx2; rabbit polyclonal antibodies by Proteintech (Wuhan, China): anti‐aP2, and anti‐β‐actin; rabbit polyclonal antibody by SAB: anti‐H3K9me2 and rabbit polyclonal antibody by Bioworld: anti‐H3K27me2. The membranes were then incubated with the corresponding horseradish peroxide‐labeled IgG (1:3000) for 2 hours. Chemiluminescence reagent (Advansta, Menlo Park, CA, USA) was finally used to visualize the results.
Chemiluminescence reagent
Manufactured by Advansta
Sourced in United States
Chemiluminescence reagent is a lab equipment product that generates light through a chemical reaction. It is used to detect and quantify specific biomolecules in various applications, such as Western blotting and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Bioworld Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti osterix, by Cell Signaling Technology (1 mentions)
Anti alp, by Cell Signaling Technology (1 mentions)
Anti c ebpa, by Cell Signaling Technology (1 mentions)
Anti c ebpα, by Proteintech (1 mentions)
Anti acc1, by Proteintech (1 mentions)
Anti pparγ, by Cell Signaling Technology (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti mdm2, by Abcam (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Anti bcl xl, by Abcam (1 mentions)
Anti p53, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti bax, by Abcam (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Anti pparγ, by Proteintech (1 mentions)
7 protocols using chemiluminescence reagent
1
Western Blot Analysis of Osteogenic Markers
2
Western Blotting of Adipogenic Markers
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Western blotting experiments were performed as previously described (23 (link)). The primary antibodies used are: rabbit monoclonal or polyclonal antibodies by Cell Signaling Technology: anti-PPARγ (#2443, 1:1000), anti-Perilipin (#9349, 1:1000), anti-C/EBPα (#8178, 1:1000) and anti-ACC1 (#4190, 1:1000); rabbit monoclonal or polyclonal antibodies by Proteintech (Wuhan, China): anti-aP2 (12802-1-AP, 1:1000), anti-FASN (10624-2-AP, 1:1000), anti-SREBP1 (66875-1-Ig, 1:2000) and anti-β-actin (66009-1-Ig, 1:5000); rabbit polyclonal antibody by Abclonal (Wuhan, China): anti-TOB2 (A7223, 1:1000). Protein bands were visualized using a chemiluminescence reagent (Advansta, Menlo Park, CA) and analyzed with Image J software.
3
Western Blotting of Cellular Proteins
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Western blotting was conducted as described [27 (link)]. After exposure to compounds and CPT for 48 h, cells were lysed by the RIPA buffer with a protease inhibitor and a phosphatase inhibitor (MCE). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into the PVDF membrane. The membranes were blocked in no-fat milk with TBST and incubated with primary antibody at 4 °C overnight. Then, the membranes were washed 3 times in TBST and incubated with secondary antibody at room temperature. After being washed, the target proteins on the membranes were detected by chemi-luminescence reagent (Advansta, San Jose, CA, USA).
4
Quantifying SMAD5 Protein Expression
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The protein expression of SMAD5 protein in transfected HT-1376 and HTB9 cells was detected by using western blot. Briefly, SMAD5 protein was extracted from the transfected BC cells using a lysis buffer containing protease and phosphatase inhibitors. The protein was separated by SDS-PAGE and then electronically transferred to a polyvinylidene fluoride membrane. The membrane was incubated with primary antibody against SMAD5 (CST, 12534S, 1 : 1000), followed by the corresponding secondary antibody (CST, 1 : 3000, 7074S). GAPDH (CST, 5174S, 1 : 1000) was applied as the control group. The resultant membrane was photographed using a chemiluminescence reagent (Advansta, USA) and analyzed with ImageJ software (BIO-RAD, USA).
5
Protein Expression Analysis in Lysed Cells
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Cell lines or tumor tissues were collected in RIPA buffer with protease inhibitor cocktails (Thermo Fisher Scientific) and lysed on ice for 30 min. Lysates were centrifuged for 15 min at 13,000× g and 4 °C, and the supernatants were collected; then, their protein concentrations were determined by the BCA Protein Assay Reagent (Thermo Fisher Scientific). Protein samples were transferred onto a PVDF membrane which was incubated with a 1:1000 dilution of primary antibodies overnight at 4 °C. Next, the membrane was incubated with HRP-conjugated rabbit or mouse secondary antibodies at room temperature for 1.5 h and developed using a chemiluminescence reagent (Advansta, San Jose, CA, USA). The commercial antibodies used in this study included anti-LPL (1:1000; Proteintech, Rosemont, IL, USA), anti-β-actin (1:5000; Cell Signaling Technology, Danvers, MA, USA), anti-p53 (1:1000; Abcam, Cambridge, UK), anti-MDM2 (1:1000; Abcam), anti-Bax (1:1000; Abcam), anti-Bcl-2 (1:1000; Abcam), and anti-Bcl-xl (1:1000; Abcam).
6
Isoform-specific shRNA and PTEN expression
7
Western Blot Analysis of Cell Lysates
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Cells were lysed and proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were incubated overnight with rabbit monoclonal anti-C/EBPα, (Abcam, Cambridge, MA), rabbit polyclonal anti-TGFBR1 (Abgent, San Diego, CA), rabbit polyclonal anti-PPARγ, anti-aP2, or anti-β-actin antibodies (Proteintech, Wuhan, China). This was then followed by incubation with the corresponding horseradish peroxide-labeled IgG (1:3000) for 1 h. Finally, chemiluminescence reagent (Advansta, Menlo Park, California) was used to visualize the results.
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