Rabbit anti erβ

Treated cells were fixed with ice-cold 100% methanol at -20°C for 15 min and washed 3 times with phosphate buffered saline (PBS) for 5 min. Non-specific binding was blocked by incubating cells in 5% BSA blocking solution (containing 10% horse serum in 1X TBS-T) for 60 min at room temperature. This was followed by washing with PBS. Thereafter, the cells were incubated with the following primary antibodies overnight at 4°C: rabbit anti-NF-B p65 (Santa Cruz; 1:100), rabbit anti-ERα (Abcam; 1:100), rabbit anti-ERβ (Santa Cruz; 1:100) and rabbit anti-MAP2 (Santa Cruz; 1:100). Following overnight incubation, cells were washed thrice with PBS and incubated for 2 h in the dark with Alexa Fluor 488conjugated donkey anti-rabbit IgG secondary antibody (Life Technologies; 1:500).
Thereafter, cells were washed with PBS and counterstained with DAPI for 5 min. After rinsing cells with PBS, excess buffer was removed and gold antifade reagent (Invitrogen) was added. All staining procedures were performed at room temperature. Fluorescence images were obtained using EVOS® FLoid® cell imaging station.

Equal amounts of protein (20 μg) were separated on a polycryamide electrophoresis gel and transferred onto a polyvinylidine fluoride (PVDF) membrane. Membranes were incubated in blocking buffer for 1 h at room temperature, washed 3 times for 10 min each in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and incubated with primary antibodies overnight at 4C. Primary antibodies used were rabbit anti-COX-2 (Santa Cruz;
1:500), rabbit anti-iNOS (Santa Cruz, 1:500), rabbit anti-IBα (Santa Cruz; 1:250), rabbit anti-phospho-IκBα (Santa Cruz, 1:250), rabbit anti-IKKα (Santa Cruz; 1:250), rabbit antiphospho-IKKα (Santa Cruz, 1:250), rabbit anti-phospho-p65 (Santa Cruz, 1:500), rabbit anti-p65 (Santa Cruz 1:500), rabbit anti-microtubule-associated protein-2 (MAP2) (Santa Cruz, 1:500) , rabbit anti-ERβ (Santa Cruz 1:250) and rabbit anti-actin (Sigma Aldrich, 1:500). Primary antibodies were diluted in TBS-T and 1% bovine serum albumin (BSA).
After extensive washing (three times for 15 min each in TBS-T), proteins were detected by incubation with Alexa Fluor 680 goat anti-rabbit secondary antibody (1:10000; Life Technologies) at room temperature for 1 h. Detection was done using a LICOR Odyssey Imager. All Western blot experiments were carried out at least three times.