Western blotting analysis using phospho-eIF2 alpha (Ser51) antibody (Cat#63068). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with phospho-eIF2 alpha (Ser51) antibody (Cat#63068, 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 phospho-eIF2 alpha (Ser51) antibody (Cat#63068). Phospho-eIF2 alpha (Ser51) expression in wild-type (WT) and EIF2S1 shRNA knockdown (KD) HeLa cells with 20 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with phospho-eIF2 alpha (Ser51) antibody (Cat#63068, 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). EIF2S1, eukaryotic translation initiation factor 2 subunit alpha.
Immunohistochemistry was performed on paraffin-embedded human sigmoid colon carcinoma using phospho-eIF2 alpha (Ser51) antibody (Cat#63068, 1:200). Antigen retrieval was done in sodium citrate buffer (pH 6.0). DAB was used for detection, with hematoxylin counterstaining. Images were acquired using a Nikon Ci-L Plus microscope (40× objective). Scale bar, 25 μm.
Flow cytometric analysis of phospho-eIF2 alpha (Ser51) expression in HT-1080 cells using phospho-eIF2 alpha (Ser51) antibody (Cat#63068, 1:2,000). Green, isotype control; red, phospho-eIF2 alpha (Ser51).
Validation of Phospho-eIF2 alpha (Ser51) knockdown using flow cytometry. Wild-type(WT, Blue) and Phospho-eIF2 alpha (Ser51) knockdown(KD, Green) HeLa cells were stained with Phospho-eIF2 alpha (Ser51) antibody(Cat#63068, 1:2,000) and analyzed using BD flow cytometer.
