Flow cytometric analysis of phospho-4E BP1 (Thr46) expression in C2C12 cells using anti-phospho-4E BP1 (Thr46) antibody (Cat#63183, 1:2,000). Green, isotype control; red, phospho-4E BP1 (Thr46).
Immunocytochemical staining of C2C12 cells with anti-Phospho-4E BP1 (Thr46) antibody (Cat#63183, 1:1,000) . Nuclei were stained blue with DAPI; Phospho-4E BP1 (Thr46) was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Medium. Scale bar, 20 μm.
Western blotting analysis using anti-phospho-4E BP1 (Thr46) antibody (Cat#63183). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with anti-phospho-4E BP1 (Thr46) antibody (Cat#63183, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Western blotting analysis using anti-phospho-4E BP1 (Thr46) antibody (Cat#63183). Phospho-4E BP1 (Thr46) expression in wild-type (WT) and EIF4EBP1 shRNA knockdown (KD) HeLa cells with 20 μg of total cell lysates. β-Tubulin serves as a loading control. The blot was incubated with anti-phospho-4E BP1 (Thr46) antibody (Cat#63183, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using NaQ™ ECL Substrate Kit (Cat#716). EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1.
Validation of 4E BP1 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with anti-Phospho-4E BP1 (Thr46) antibody (Cat#63183, 1:2,000) and analyzed using BD flow cytometer.
Immunocytochemical staining of HeLa cells using anti-Phospho-4E BP1 (Thr46) antibody (Cat#63183, 1:1,000), Top panel: wild-type (WT); Bottom panal: Phospho-4E BP1 (Thr46) shRNA knockdown (KD). Nuclei were stained blue with DAPI; Acyl-CoA dehydrogenase short chain was stained magenta with Alexa Fluor® 647. Scale bar, 20 μm.