|Detection of Human Phospho‑Insulin R (Y1162/Y1163) and Phospho‑IGF‑I R (Y1135/1136) by Western Blot. Western blot shows lysates of A431 human epithelial carcinoma cell line untreated (-) or treated (+) with 100 μM pervanadate (PV) for 10 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human Phospho-Insulin R (Y1162/Y1163)/IGF‑I R (Y1135/Y1136) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2507), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for Phospho-Insulin R (Y1162/Y1163) at 95 kDa and Phospho-IGF‑I R (Y1135/1136) at approximately 100 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
|Phospho‑Insulin R (Y1162/1163)/IGF-I R (Y1135/1136) in A431 Human Cell Line. Insulin R phosphorylated at Y1162/1163 and IGF‑I R phsophorylated at Y1135/1136 were detected in immersion fixed A431 human epithelial carcinoma cell line untreated (lower panel) or treated (upper panel) with pervanadate using Rabbit Anti-Human Phospho-Insulin R (Y1162/1163)/IGF-I R (Y1135/1136) Cross-reactive Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2507) 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). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
Intracellular Staining by Flow Cytometry
|Detection of Insulin R/CD220 in HeLa Human Cell Line by Flow Cytometry. HeLa human cervical epithelial carcinoma cell line was unstimulated (light orange open histogram) or treated with 100 μM pervanadate for 15 minutes, then stained with Rabbit Anti-Human Phospho-Insulin R (Y1162/1163)/IGF‑I R (Y1135/1136) cross-reactive Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF2507, dark orange filled histogram) or isotype control antibody (Catalog # AB-105-C, blue open histogram), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.|
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