The cells were lysed using mammalian protein extraction reagent radioimmunoprecipitation assay lysis buffer (RIPA; Beyotime, Haimen, China), supplemented with protease inhibitor cocktail (Roche) and PMSF (Roche). 50 μg of the protein extractions was separated by 10% SDS-PAGE, transferred to 0.22-mm nitrocellulose (NC) membranes (Sigma-Aldrich), and incubated with specific antibodies. The autoradiograms were quantified by densitometry (Quantity One software). Anti-FOXA1 (ab23738), Anti-BIK (ab52182), and Anti-XAF1 (ab17204) were from Abcam. Results were normalized to the expression of GAPDH.
0.22 mm nitrocellulose nc membranes
Manufactured by Merck Group
The 0.22-mm nitrocellulose (NC) membranes are thin, porous sheets made of nitrocellulose material. These membranes are designed to facilitate the filtration and separation of materials, particularly small particles or molecules, from liquid or gas samples.
Automatically generated - may contain errors
Lab products found in correlation
Radioimmunoprecipitation assay (ripa), by Beyotime (6 mentions)
Quantity one software, by Bio-Rad (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (4 mentions)
Protease inhibitors cocktail, by Roche (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Anti klf2, by Merck Group (3 mentions)
Anti foxa1 ab23738, by Abcam (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Anti ezh2, by Abcam (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3b, by Cell Signaling Technology (1 mentions)
Anti gsk3b, by Cell Signaling Technology (1 mentions)
Anti hoxb7, by Abcam (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
Anti p21, by Abcam (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
7 protocols using 0.22 mm nitrocellulose nc membranes
1
Western Blot Analysis of Protein Markers
2
Western Blot Analysis of KLF2
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The cells were lysed by using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche). Fifty micrograms of the protein extractions were separated by 10 % SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies. The autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Anti-KLF2 was purchased from Sigma (1:1000). Results were normalized to the expression β-actin (Mouse anti-β-actin) (Sigma (1:1000)).
3
Western Blot Analysis of Signaling Pathways
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The cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche) and PMSF (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies.The autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Anti-EZH2 was from Abcam (Hong Kong, China). Anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-phospho-GSK3b and anti-GSK3b were from Cell Signaling Technology (Boston, MA, USA). Anti-HOXB7 was from Abcam. Anti-p53 was from Santa Cruz Biotechnology (Dallas, TX, USA). Results were normalized to the expression of GAPDH (Rabbit anti-GAPDH).
4
Quantifying p21 Protein Expression
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The cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche) and PMSF (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies.The autoradiograms were quantified by densitometry (Quantity One software; Anti-p21 was from Abcam. Results were normalized to the expression of GAPDH.
5
Quantifying KLF2 Protein Expression
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Cells were lysed by using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche). Fifty micrograms of the protein extractions were separated by 10 % SDS-PAGE transferred to 0.22-mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies. The autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA). Anti-KLF2 was purchased from Sigma (1:1000). Results were normalized to the expression GAPDH (mouse anti-GAPDH) (Sigma (1:1000)).
6
Western blot analysis of KLF2
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Cells were lysed by using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitor cocktail (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22-mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies. The autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA). Anti-KLF2 was purchased from Sigma (1:1,000). Results were normalized to the expression GAPDH (mouse anti-GAPDH) (Sigma; 1:1,000).
7
Quantitative Protein Analysis of Transfected Cells
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After 48 h of transfection, cells were lysed using a lysis buffer which contained the mammalian protein extraction reagent RIPA (Beyotime, Beijing, China), a protease inhibitor cocktail (Roche, Basel, Switzerland), and phenylmethylsulfonyl fluoride (PMSF) (Roche). Cell protein lysates that contained 50-µg protein were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto 0.22-mm nitrocellulose (NC) membranes (Sigma-Aldrich), and incubated with specific antibodies. Specific bands were detected by enhanced chemiluminescence (ECL) chromogenic substrate and quantified by densitometry (Quantity One software; Bio-Rad Laboratories, Hercules, CA, USA). GAPDH antibody was used as control; anti-EZH2, MMP2, and MMP9 (1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
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