Detection of Mouse, Rat, and Human Phospho-ERK1 (T202/Y204) and ERK2 (T185/Y187) by Western Blot. Western blot shows lysates of NIH‑3T3 mouse embryonic fibroblast cell line untreated (-) or treated (+) with 100 ng/mL Rabbit Anti-Human PDGF (Catalog # 120-HD) and PC‑12 rat adrenal pheochromocytoma cell line untreated or treated with 100 ng/mL Recombinant Rat beta -NGF (Catalog #|
556‑NG) and HeLa human cervical epithelial carcinoma cell line untreated or treated with 200 nM PMA. PVDF membrane was probed with 0.5 µg/mL of Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Monoclonal Antibody (MAB1018), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for Phospho-ERK1 (T202/Y204)/ ERK2 (T185/Y187) at approximately 40-45 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 3.
|Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) in HeLa Human Cell Line. ERK1/ERK2 phosphorylated at T202/Y204 (ERK1) and T185/Y187 (ERK2) was detected in immersion fixed Hela human cervical epithelial carcinoma cells, unstimulated (lower panel) and stimulated (upper panel) with PMA using Rabbit Anti-Human/Mouse/Rat Phospho-ERK1/ERK2 (ERK1 T202/Y204, ERK2 T185/Y187) Monoclonal Antibody (Catalog # MAB1018) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
Intracellular Staining by Flow Cytometry
|Detection of Phospho-ERK1/ERK2 in PMA-treated Jurkat Human Cell Line by Flow Cytometry. Jurkat human acute T cell leukemia cell line were unstimulated (light orange open histogram) or treated with 50 ug/mL PMA for 10 minutes (dark orange filled histogram), then stained with Rabbit Anti-Human/Mouse/Rat Phospho-ERK1/ERK2 (ERK1 T202/Y204, ERK2 T185/Y187) Monoclonal Antibody (Catalog # MAB1018) or control antibody (Catalog # AB‑105‑C, blue open histogram), followed by Fluorescein-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0112). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with ice-cold methanol.|
|Detection of Human Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) by Simple WesternTM. Simple Western lane view shows lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 200 nM PMA and Ionomycin for 20 minutes, loaded at 0.2 mg/mL. A specific band was detected for Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) at approximately 42 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-ERK1/ERK2 (ERK1 T202/Y204, ERK2 T185/Y187) Monoclonal Antibody (Catalog # MAB1018). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.|
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