All lanes : Anti-eIF4EBP1 (phospho T46) antibody [19HCLC] (ab277767) at 2 µg/ml

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : HEK-293 whole cell lysate treated for 10 minutes with 100 nM/mL of EGF
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : MCF7 whole cell lysate treated for 15 minutes with 150 µg/mL of Insulin

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG - HRP at 1/5000 dilution

Predicted band size: 13 kDa

Immunofluorescent analysis of Phospho-4EBP1 pThr46 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells using ab277767 followed by detection using an Alexa Fluor^® 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor^® 594 phalloidin (red) (image C). Image D is a composite image showing nuclear localization of phosphorylated 4EBP1 and Image E is a composite image of cells showing inhibition of antibody binding after competition with the phosphorylated peptide.

All lanes : Anti-Cx36 antibody [2HCLC] (ab277766) at 2 µg/ml

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
treated with EGF
Lane 2 : Preincubation with the phosphopeptide, HEK-293 whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate treated with PDGF
Lane 4 : Preincubation with the phosphopeptide, NIH/3T3 whole cell lysate

Predicted band size: 13 kDa

Immunofluorescent analysis of 4EBP1 (pT46) was done on 70% confluent log phase HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ab277767 at a dilution of 2 μg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor^® 488 Goat anti-Rabbit IgG Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor^® 594 Phalloidin. Panel d is a merged image showing nuclear localization of 4EBP1 (pT46). Panel e shows competition with 4EBP1 (pT46) peptide. The images were captured at 20X magnification.