Rabbit anti pias1

MDA-MB-231 cells transfected with green fluorescence protein (GFP)-expressing control or PIAS1 RNAi plasmids were seeded in 96-well plate at 105 cell/ ml. Two days later, cells were 4% paraformaldehyde-fixed (Fisher), permeabilized with 0.2% Triton-X (Sigma), blocked using 5% bovine serum albumin (Roche) and 5% calf serum (VWR) containing PBS. Cells were then subjected to indirect immunocytochemistry for PIAS1 using rabbit-anti PIAS1 (Epitomics), as primary antibody, and a Cy3-conjugated anti-rabbit IgG (Jackson Laboratories), as secondary antibody, and Hoechst-DNA-staining using bisBenzimide H33342 trihydrochloride (Invitrogen to visualize their nuclei. Fluorescence images were acquired using the Cellomics Kinetic Scan Reader that is equipped with Carl Zeiss Axiom x microscope and a charge coupled device (CCD) digital camera [18 (link), 35 (link)]. The spot detector bioapplication was used to quantify PIAS1 fluorescence intensity where each cell was identified by nuclear stain and GFP signal (transfected cells). Each condition was carried out in triplicates, and the immunofluorescence data were averaged from a minimum of 2000 cells per well. The intensity of PIAS1 immunofluorescence in transfected MDA-MB-231 cells was expressed as a percent of the PIAS1 immunofluorescence in the control vector-transfected cells (Figure S3B).

Cell were incubated for 10 minutes at 4°C in TNTE lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Triton-X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μg/ ml pepstatin A (Sigma), 100 μg/ml benzamidine chloride (Calbiochem), and 1 mg/ml trypsin inhibitor, 10 μg/ ml antipain, 50 μg/ml aprotinin and 10 μg/ml leupeptin (Roche Applied Biosciences)), and phosphatase inhibitors (10 mM sodium pyrophosphate and 25 mM sodium fluoride (EM Sciences), and 1 mM sodium orthovanadate (Alfa Aesar)). Lysates were centrifuged at 15,000X g for 10 minutes at 4°C, and small aliquots were subjected to protein concentration determination using Bradford-based protein assays (Bio-Rad Laboratories). Cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose membrane (Bio-Rad Laboratory). The blots were incubated with mouse anti-FLAG (Sigma), rabbit anti-PIAS1 (Epitomics) or rabbit anti-actin (Sigma), as the primary antibody and HRP-conjugated donkey anti-mouse or anti-rabbit IgG (Jackson Laboratories) as secondary antibodies, followed by ECL and signal detection using a VersaDoc 5000 Imager (Bio-Rad Laboratories). Densitometry was performed using Quantity One software (Bio-Rad Laboratories).