Protein was extracted using RIPA buffer (Sangon Biotech, Shanghai, People’s Republic of China) containing proteinase inhibitors (MedChem Express) and phosphatase inhibitors (MedChem Express). The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). The following antibodies were used for Western blot: anti-CDK7 (Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (Cell Signaling Technology), anti-poly(ADP-ribose) polymerase (anti-PARP) (Cell Signaling Technology), anti-cleaved PARP (Cell Signaling Technology), anti-RNA Pol II (Abcam, Cambridge, UK), anti-phospho RNA Pol II (Ser2) (Abcam), anti-phospho RNA Pol II (Ser5) (Abcam), anti-phospho RNA Pol II (Ser7) (Millipore), anti-CDK1 (Abcam), and anti-CDK1 (phospho T161) (Abcam).
Proteinase inhibitor
Manufactured by MedChemExpress
Sourced in United States
Proteinase inhibitor is a laboratory reagent used to prevent the degradation of proteins by proteolytic enzymes. It functions by inhibiting the activity of proteases, enzymes that break down proteins. This preserves the integrity and structure of proteins during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by MedChemExpress (3 mentions)
Gapdh, by BioLegend (2 mentions)
Anti cdk7, by Cell Signaling Technology (1 mentions)
Anti poly adp ribose polymerase anti parp, by Cell Signaling Technology (1 mentions)
Anti phospho rna pol 2 ser2, by Abcam (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti cdk1, by Abcam (1 mentions)
Ripa buffer, by Sangon (1 mentions)
Anti rna pol 2, by Abcam (1 mentions)
Phospho erk p erk, by Cell Signaling Technology (1 mentions)
Runx2, by Novus Biologicals (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Extracellular signal regulated kinase erk, by Cell Signaling Technology (1 mentions)
C jun n terminal kinase jnk, by Cell Signaling Technology (1 mentions)
Lamin b1, by Santa Cruz Biotechnology (1 mentions)
Peroxisome proliferator activated receptor gamma pparγ, by Cell Signaling Technology (1 mentions)
5 protocols using proteinase inhibitor
1
Western Blot Analysis of Cell Signaling Pathways
2
Quantitative Western Blot Analysis
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RD cells were harvested and lysed in Radioimmunoprecipitation assay buffer (RIPA buffer) with a mixture of proteinase inhibitors (MedChem Express, Monmouth Junction, NJ, USA). The concentration of proteins was quantified by bicinchoninic acid protein assay (Biosharp, PR China) and equal amounts of proteins were loaded and separated by 10 % SDS polyacrylamide gels, and then transferred to a nitrocellulose membrane. The membrane was blocked with blocking buffer (0.1 % Tween-20 in PBS containing 5 % milk) and then was incubated overnight with mouse anti-EIF4G antibody diluted in blocking buffer at 4 °C. The membrane was then washed three times in 0.1 % Tween-20/TBS and incubated with anti-mouse IgG conjugated to AlexaFluor790 (Jackson Immuno Research, West Grove, PA, USA) for 2 h at room temperature. The immunoblots were visualized using an Odyssey Fc Imager (Lincoln, NE, USA).
3
Protein Extraction and Immunoblotting Analysis
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Cytoplasmic and nuclear proteins were extracted by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™) with proteinase inhibitor (MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (MedChemExpress) [44 (link)]. The cell lysates were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with the following indicated antibodies: bone morphogenetic protein-4 (BMP4; Cusabio Life Science, Wuhan, China), runx family transcription factor 2 (Runx2; Novus Biologicals, Centennial, Colorado, USA), ALP (R&D Systems, Minneapolis, MN, USA), extracellular signal-regulated kinase (ERK; Cell Signaling Technology), phospho-ERK (p-ERK; Cell Signaling Technology), c-Jun N-terminal kinase (JNK; Cell Signaling Technology), p-JNK (Cell Signaling Technology), p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), β-catenin (Cell Signaling Technology), Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (BioLegend, SanDiego, CA, USA). GAPDH was used as a housekeeping gene for normalization. Lamin B1 was used for normalization of nuclear β-catenin.
4
Adipocyte Differentiation Protein Analysis
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The proteins were extracted from the fully differentiated 3T3-L1 cells using passive lysis buffer (Promega Corporation, Fitchburg, WI, USA) supplemented with a proteinase inhibitor (MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (MedChemExpress), referring to the manufacturer's descriptions, and were quantified by BCA assay kit (Life Technologies). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies: peroxisome proliferator-activated receptor gamma (PPARγ; dilution 1 : 1000; Cell Signaling Technology, Beverly, MA, USA), CCAAT/enhancer-binding protein alpha (C/EBPα; dilution 1 : 1000; Cell Signaling Technology), fatty acid-binding protein 4 (FABP4; dilution 1 : 1000; Cell Signaling Technology), fatty acid synthase (FAS; dilution 1 : 1000; Cell Signaling Technology), protein kinase B (AKT; dilution 1 : 1000; Bioss, Woburn, MA, USA), phospho-Akt (p-AKT; dilution 1 : 2000; Cell Signaling Technology), tumor necrosis factor-alpha (TNF-α; dilution 1 : 1000; Cusabio Life Science, Wuhan, China), and GAPDH (dilution 1 : 2000; BioLegend, San Diego, CA, USA). GAPDH was used to achieve equal loading of protein. The protein signals on the membranes were developed using ECL western blotting substrate (Daeil Lab Service).
5
Quantitative Western Blot Analysis
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Whole-cell protein lysates were extracted with SDS protein lysis buffer with proteinase inhibitor (MedChemExpress) and protein concentration determined by standard Bradford protein assay (Bio-Rad) at 590 nm using the microplate reader (Labexim Products, University Research Facility in Life Sciences of The Hong Kong Polytechnic University). Twenty micrograms of protein were loaded onto SDS-PAGE gels and transferred onto PVDF membrane after electrophoresis. After 1 h blocking in 5% skim milk and incubation of primary antibody (1:2000) at 4 °C overnight, the membrane was incubated with secondary antibody (anti-rabbit or anti-mouse antibody, 1:2000) at room temperature for 1 h. The membrane was washed 3 times with 1X TBST at 5-min intervals before and after secondary antibody incubation. Clarity Western ECL Substrate (Bio-Rad) was used for detection. Primary antibody dilution ratios: Merlin (D1D8) rabbit mAb (Cell Signaling Technologies, CST), non-phospho (active) β-catenin (Ser33/37/Thr41) (D13A1) rabbit mAb (CST), β-catenin (D10A8) rabbit mAb (CST), MYC (CST) and β-actin mouse mAb (ABclonal). Semi-quantitative analyses of protein bands were done using ImageJ software. Intensity of bands was calculated as an arbitrary value relative to ACTB expression level.
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