Whole-cell lysates (WCLs) were generated in modified radioimmunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and 0.1% SDS, without sodium deoxycholate), supplemented with protease (40 µg/ml PMSF, 2 µg/ml antipain, 2 µg/ml pepstatin A, 20 µg/ml leupeptin, and 20 µg/ml aprotinin) and phosphatase (10 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate, and 10 mM sodium pyrophosphate) inhibitors. After clarification of debris by a microfuge, samples were quantified with the DC Protein Assay Kit (Bio-Rad). Total lysate protein was resolved by standard SDS-PAGE and transferred in 1× transfer buffer and 15% methanol. Membranes were incubated with their respective primary and secondary antibodies labeled with IRDye (680 and 800 nm) and then visualized using the LI-COR system. Antibodies against phospho-p42/44 MAPK (rabbit polyclonal; #9101; 1:1,000) were obtained from Cell Signaling. Monoclonal pan-RAS antibody (clone Ab-3; OP40-100UG; 1:1,000) was obtained from Millipore. Rabbit polyclonal antibodies against SHP2 (sc-280; 1:1,000) and mouse monoclonal ERK-2 (D2: sc-1647; 1:1,000) were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-SOS1 (MA5-17234) was purchased from Invitrogen. Rabbit monoclonal anti-DUSP6 antibody EPR129Y was obtained from Abcam (#ab76310).
Epr129y
Manufactured by Abcam
EPR129Y is a monoclonal antibody that recognizes the human EGFR protein. It can be used in various immunoassay applications to detect and quantify EGFR expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti phospho erk, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
She78 7, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto substrate kit, by Thermo Fisher Scientific (1 mentions)
Epr8185, by Abcam (1 mentions)
Pierce coomassie plus protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
4 protocols using epr129y
1
Whole-Cell Lysate Preparation and Immunoblotting
2
FGF-2 Stimulation of Human Chondrocytes
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P0–P3 HACs were cultured as monolayers and transfected 36–48 h before lysis. When appropriate, HACs were stimulated with a 25 nM human FGF-2 (Prepotech, UK). The age and gender of the HACs donors used for each experiment are indicated in the figure legends. HACs were lysed in a radio-immunoprecipitation assay buffer (RIPA—150 mM NaCl, 10 mM Tris, pH 7.2, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid) and 10–30 μg total protein extract subjected to SDS-polyacrilamide gel electrophoresis and transferred to PVDF membranes (Merck-Millipore, Germany). Mouse monoclonal anti-Ago2 (clone2E12-1C9, Abnova, 1:1000 dilution), rabbit monoclonal anti-DUSP6 (EPR129Y, Abcam, 1:1000 dilution), rabbit monoclonal anti-phospho-ERK (Cat No. 05-797R, Sigma-Aldrich, 1:1000 dilution), rabbit polyclonal anti-ERK (Cat No. sc-292838, SantaCruz Biotechnologies, 1:500 dilution), mouse monoclonal anti-Tubulin (clone B-5-1-2, Sigma-Aldrich, 1:5000 dilution), HRP-conjugated anti-mouse IgG secondary (Cat No. NA931, GE Healthcare, 1:5000 dilution), and HRP-conjugated anti-rabbit IgG secondary (Cat No. NA934, GE Healthcare, 1:5000 dilution), antibodies were used. Proteins were visualized by ECL fluorography (GE Healthcare, Germany).
3
Western Blot Analysis of Signaling Pathways
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Total cellular proteins were extracted at 4 °C using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins (20 µg) were resolved on 8–12% SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Western blots were probed with antibodies against p-ERK (Tyr204, 1:2000; E4; Santa Cruz, CA), total ERK (1:2000; 137F5; Cell Signalling, Danvers, MA), p-AKT (Ser473, 1:1000, 736E11, Cell Signalling), total AKT (1:1000; 40D4, Cell Signalling), p-S6 (Ser235/236, 1:2000, 2F9, Cell Signalling), p-RSK1/2/4 (Ser363, 1:2000, Santa Cruz), total PI3K (1:1000, C73F8, Cell Signalling), HA-tag (1:1000, 6E2, Cell Signalling), DUSP6 (1:1000, EPR129Y, Abcam), EGF receptor (1:1000, D38B1, Cell Signalling), p-EGF receptor (Tyr1068, 1:500, Cell Signalling), MITF (1:1000, C5, Calbiochem), SOX10 (1:500, N-20, Santa Cruz), E-cadherin (1:1000, SHE78-7, Invitrogen), NRas (1:1000, F155, Santa Cruz) and ß-Actin (1:3000, I-19, Santa Cruz). Each western blot was performed using at least two biological replicates. For cell transduction work we performed two biological replicates from each of two independent transductions.
4
Protein Expression Analysis by Western Blot
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Cells were lysed on ice using RIPA buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1% triton X-100, 0.1% sodium deoxycholate and 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktails (Roche). Protein extracts were clarified and concentrations were measured with Pierce Coomassie Plus Protein Assay reagent (Thermo Fisher Scientific). Lysates were then resolved on SDS-PAGE gels (BioRad), and transferred to Nitrocellulose blots. Membranes were probed with primary antibodies against AXIN1 (2087, CST), TCF4 (2569, CST) pFAK Y397 (SAB4504403, Sigma), DUSP6 (EPR129Y, Abcam), SPRY2 (EPR4318(2)(B), Abcam), pERK (4370, CST), tERK (4696, CST), Vinculin (EPR8185, Abcam), and β-tubulin (E7, DSHB). Blots were subsequently incubated with HRP conjugated secondary antibodies. For signal development, Supersignal West Femto Substrate kit (Thermo Fisher Scientific) was used, followed by image acquisition using darkroom development. ImageJ was used for band intensity quantitation.
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