For protein-based assays, 1.0 × 106 cells/well were seeded in 6-well plates one day before treatment (seeding volume 2 mL/well). 1000X stock solutions of the indicated drugs, or the corresponding volume of DMSO, were added to each well 24 h post-seeding. Drug-treated cells were incubated for the indicated times, mostly 24 h. Total proteins were extracted with RIPA buffer (Pierce, Appleton, WI, USA), supplemented with protease-inhibitors and phosphatase-inhibitors cocktails (Sigma, San Francisco, CA, USA). 30 μg of whole cell lysates were separated on 4–12% Bis-Tris gels and transferred to 0.45 μm nitrocellulose membranes (Invitrogen). Protein targets of interest were detected using the following antibodies: AMOTL2, Proteintech Cat. No. 23351-1-AP; CTGF, Cell-Signaling #86641; CYR61, Cell-Signaling #14479; YAP1, Cell-Signaling #14074; TEAD1, Cell-Signaling #12292; Pan-TEAD, Cell-Signaling #13295; Cleaved-PARP1, Cell-Signaling #5625; Cleaved-CASP3, Cell-Signaling #9579.
Protease inhibitors and phosphatase inhibitors cocktails
Manufactured by Merck Group
Sourced in United States
Protease inhibitors and phosphatase inhibitors cocktails are laboratory reagents used to prevent the degradation of proteins in biological samples. They contain a mixture of compounds that inhibit the activity of proteases and phosphatases, enzymes that can break down or modify proteins. These cocktails are commonly used in protein extraction, purification, and analysis procedures to preserve the integrity and stability of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
0.45 μm nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Ab115522, by Abcam (1 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Nb100 266, by Novus Biologicals (1 mentions)
A5441, by Merck Group (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Mouse anti vinculin, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Guinea pig anti insulin, by Fortrea (1 mentions)
3 protocols using protease inhibitors and phosphatase inhibitors cocktails
1
Protein Expression Analysis in Cells
2
Quantitative Analysis of DNA Methyltransferases
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The frozen tissue was re-suspended in passive lysis buffer (Promega Corporation, Madison, WI, USA) including freshly added protease inhibitors and phosphatase inhibitors cocktails (Sigma-Aldrich, Darmstadt, Germany). Protein concentrations in the supernatant fractions were determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Protein lysates were separated with 8 or 12% SDS-acrylamide gels, and transferred onto nitrocellulose membranes. After blocking for 60 min in a 5% skim milk solution, membranes were incubated overnight at 4°C with monoclonal rabbit anti-DNMT1 antibody at 1:1,000 (D59A4, Cell Signaling Technology, Inc., Danvers, MA, USA), monoclonal rabbit anti-DNMT3A at 1:1,000 (D23G1, Cell Signaling Technology, Inc.) and polyclonal rabbit anti-DNMT3B at 1:1,000 (NB100-266, Novus Biologicals, LLC), polyclonal rabbit anti-DNMT3L at 1:1,000 (ab115522, Abcam) or monoclonal mouse anti-β-actin at 1:5,000 (A5441, Sigma-Aldrich). Signals from horseradish-peroxidase-conjugated secondary antibodies were visualized by enhanced chemilluminescence solution (GE Healthcare Life Sciences, Chalfont, UK) and processed with an UVP visualizer (UVP, Inc., Upland, CA, USA). Quantification was performed using VisionWorks LS version 7.0 from the UVP visualizer itself. Experiments were repeated in triplicate.
3
Islet Protein Extraction and Western Blotting
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Hand-picked islets were washed once with PBS and then resuspended in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors cocktails (Sigma) as described32 (link). Islets were lysed by repeated pipetting or triturating through a 30G syringe needle. Lysates were resolved on a 4–12% NuPAGE Bis-Tris gel, and then electrotransferred to nitrocellulose membrane for western blotting. Antibodies used were mouse anti-proinsulin (ALPCO), guinea pig anti-insulin (Covance), and mouse anti-vinculin (Millipore). Horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc., with protein visualized by ECL (Millipore).
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