All lanes : Anti-Rb (phospho S807 + S811) antibody [13HCLC] (ab277774) at 2 µg/ml

Lane 1 : LNCaP (Human prostate cancer cell line) whole cell extract
Lane 2 : LNCaP (Human prostate cancer cell line) whole cell extract (cells treated with 83 nM Nocadazole for 24 hours)
Lane 3 : K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell extract
Lane 4 : K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell extract (cells treated with 83 nM Nocadazole for 24 hours)
Lane 5 : MCF-7 (human breast adenocarcinoma cell line) whole cell extract
Lane 6 : MCF-7 (human breast adenocarcinoma cell line) whole cell extract (cells treated with 83 nM Nocadazole for 24 hours)

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

Predicted band size: 106 kDa

Western blot analysis was performed on Nuclear enriched extracts (30 μg lysate) of LNCaP (human prostate cancer cell line) (Lane 1), LNCaP (human prostate cancer cell line) treated with Nocodazole (83 nM for 24hrs) (Lane 2), K562(human chronic myelogenous leukemia lymphoblast cell line) (Lane 3), K562(human chronic myelogenous leukemia lymphoblast cell line) treated with Nocodazole (83 nM for 24hrs) (Lane 4), MCF7 (human breast adenocarcinoma cell line) (Lane 5) and MCF7 (human breast adenocarcinoma cell line) treated with Nocodazole (83 nM for 24hrs) (Lane 6). The blots were probed with Anti-Rb (pS 807/811) Recombinant Rabbit Multiclonal Antibody (ab277774, 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 110 kDa band corresponding to Rb (pS 807/811) was observed across the cell lines tested and * non specific bands also observed. 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 wet transfer method. 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, LNCaP cells were fixed and permeabilized for detection of endogenous Rb (pS 807/811) using Rb (pS 807/811) Recombinant Rabbit Multiclonal Antibody (ab277774, 2 μg/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 Rb (pS 807/811) 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 Rb (pS 807/811). Panel e) shows loss of signal by competition with the Rb (pS 807/811) phosphopeptide, demonstrating antibody specificity, and panel f) demonstrates no competition with the non-phospho peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.