All lanes : Anti-JunB antibody (ab277769) at 2 µg/ml

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 3 : A431 (human epidermoid carcinoma cell line) whole cell lysate
Lane 4 : PC-3 whole cell lysate
Lane 5 : K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate

Secondary
All lanes : Goat Anti_rabbit HRP conjugate at 0.4 µg/ml

Predicted band size: 36 kDa

Western blot analysis was performed on whole cell extracts (30 μg lysate) of HeLa (human epithelial cell line from cervix adenocarcinoma) (Lane1), U-87MG (Lane 2), A431 (human epidermoid carcinoma cell line) (Lane 3), PC3 (human prostate adenocarcinoma cell line) (Lane 4) and K562 (human chronic myelogenous leukemia lymphoblast cell line) (Lane 5). The blots were probed with Anti-Jun B Recombinant Rabbit Multiclonal Antibody (ab277769, 1-2 μg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (0.4 μg/mL, 1/2500 dilution). A 36 kDa band corresponding to Jun B was observed across cell lines tested. Resolved proteins were 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 ECL Western blotting Substrate.

All lanes : Anti-JunB antibody (ab277769) at 2 µg/ml

Lane 1 : HeLa (untransfected) whole cell lysate
Lane 2 : HeLa (transfected with scrambled siRNA) whole cell lysate
Lane 3 : HeLa (transfected with target-specific siRNA) whole cell lysate

Predicted band size: 36 kDa

Knockdown of Jun B was achieved by transfecting HeLa (human epithelial cell line from cervix adenocarcinoma) cells with Jun B specific siRNAs (Silencer^® select ). Western blot analysis (Fig a) was performed using whole cell lysates from the Jun B knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Jun B Recombinant Rabbit Multiclonal Antibody (ab277769, 1-2 μg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, HRP conjugate (0.4 μg/mL, 1/2500 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Jun B.

Immunofluorescence was performed on fixed and permeabilized HeLa cells for detection of JunB using Anti-Jun B Recombinant Rabbit Multiclonal Antibody (ab277769, 1 μ/mL) and labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Panel a) shows representative cells that were stained for detection and localization of JunB protein (green), Panel b) is stained for nuclei (blue)  with DAPI. Panel c) represents cytoskeletal F-actin staining using Alexa Fluor^® 555 Rhodamine Phalloidin (1/300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of JunB Panel e) represents control cells with no primary Antibody to assess background.

Chromatin Immunoprecipitation (ChIP) was performed using Anti-JunB Recombinant Rabbit Multiclonal Antibody (ab277769, 5 μg) on sheared chromatin from 2 million HeLa cells. Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system with optimized PCR primer pairs for the promoters of the active MMP13, CREB5, JAG1 region used as positive control target gene, and the region of the inactive SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.