RNA was isolated from cell lines (RNeasy® Mini, Qiagen, Valencia, CA, USA) and cDNA was generated (iScript, Bio-Rad, Hercules, CA, USA). qRT-PCR was performed using validated Taqman primers (CXCR4, 18S and B2M; Life Technologies, Grand Island, NY, USA). Analysis was performed in triplicate using the LightCycler® 480 System (Roche Applied Science, Indianapolis, IN, USA) and average Cp values were normalized relative to reference genes (18S and B2M) within each sample using ΔΔ Cp method. Levels of phospho-ERK (Cell Signaling, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370), phospho-Akt (Cell Signaling, Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060), Akt (Cell Signaling, Akt (pan) (C67E7 Rabbit mAb #4691), Erk (Cell Signaling, p44/42 MAPK (Erk 1/2) #9102) and ACTIN (Abcam, Anti-beta Actin antibody (HRP) (ab20272)) were determined in whole cell lysates using standard western blot assays as previously described [15 (link)].
Phospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e xp rabbit mab 4370
Manufactured by Cell Signaling Technology
The Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 is a monoclonal antibody that recognizes endogenous levels of p44 and p42 MAP kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204.
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2 protocols using phospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e xp rabbit mab 4370
1
Quantitative mRNA and Protein Expression Analysis
2
Protein Extraction and Immunoblotting
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Total membrane and phosphorylated proteins were extracted from Sca-1+ cells. Samples (30 μg) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. Membranes were incubated with the indicated primary antibody and then with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 2000). Antibodies included anti-P2Y2R (extracellular) (1:200, Alomone), anti-P2Y6R (extracellular) (1:400, Alomone), anti-caveolin-1 (D46G3 XP®Rabbit mAb, 1:1000, Cell Signalling Technology), phospho-p44/42MAPK(Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP®RabbitmAb#4370 (1:2000, Cell Signalling Technology) and p44/42 MAPK (Erk1/2) (137F5) rabbit mAb (1:1000, Cell Signalling Technology). Refer to the online Supplemental Methods 1.9 for detailed methods.
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