All lanes : Anti-ATF2 (phospho Y69 + T71) antibody [24HCLC] (ab277776) at 2 µg/ml

Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell extract
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) (treated with 25 ug/ml Anisomycin for 30 minutes) whole cell extract
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell extract
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) (treated with 25 ug/ml Anisomycin for 30 minutes) whole cell extract

Secondary
All lanes : Goat Anti-Rabbit HRP conjugate at 0.4 µg/ml

Predicted band size: 55 kDa

Western blot analysis was performed on Nuclear enriched extracts (30 μg lysate) of NIH/3T3 (Lane 1), NIH/3T3 treated with Anisomycin (25 μg/mL for 30mins) (Lane 2), HeLa (human epithelial cell line from cervix adenocarcinoma) (Lane 3) and HeLa (human epithelial cell line from cervix adenocarcinoma) treated with Anisomycin (25 μg/mL for 30mins) (Lane 4). The blots were probed with Anti-ATF2 (pT69/71) Recombinant Rabbit Multiclonal Antibody (ab277776, 1-2 μg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, HRP conjugate (0.4 μg/mL, 1/2500 dilution). A 55 kDa band corresponding to ATF2 (pT69/71) was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex^®NuPAGE4-12% Bis-Tris gel, XCell SureLock^™ Electrophoresis System and Novex^® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce^™ ECL Western blotting Substrate.

For immunofluorescence analysis, PANC-1 cells were fixed and permeabilized for detection of endogenous ATF2 pT69/71 using ATF2 (pT69/71) Recombinant Rabbit Multiclonal Antibody (ab277776, 2 μ/mL) and labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Nuclei (blue) were stained with DAPI, and Alexa Fluor^® 555 Rhodamine Phalloidin (1/300) was used for cytoskeletal F-actin (red) staining. Detection and localization of ATF2 pT69/71 (green) in the nucleus can be clearly observed in cells treated with Insulin (100 nM, 10 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the ATF2 pT69/71 peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.