Anti p53 mab

Frozen tissues or cell pellets were treated with RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, 1 mM EGTA, 1% Triton X-100, 0.1% SDS and 1% sodium deoxy cholate, pH 8.0) in the presence of a protease inhibitors cocktail (Sigma). Equal amounts of total protein (25 μg or otherwise stated) were subject to SDS-PAGE (8%). Antibodies used in this study included the following: mouse anti-STIM1 mAb (1:1000, BD Transduction Laboratories, Clone 44); rabbit anti-Orai1 pAb (1:1500, Millipore, against residues 22-40 of the human protein); anti-Tubulin pAb (1:1000, Abcam, against residues 1-100 of the human protein); mouse anti–E-catenin (1:4000, Sigma); rabbit anti-Cdc2 mAb (1:1000, Abcam, #ab32384); mouse anti-Vimentin mAb (1:500, Millipore, # CBL202); anti-cyclinB1 mAb (1:1000, Abcam, #ab32053); anti-p53 mAb (1:1000, Santa.Cruz); rabbit anti-p27 pAb (1:1000, Cell Signaling Technology); rabbit anti-Rac1 pAb (1:500, Proteintech); mouse anti–ß-Actin (1:4000; Sigma); and horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL). The chemiluminescence of proteins transferred to PVDF membranes was detected with ECL Plus (GE Healthcare Amersham, Piscataway, NJ). Relative protein expression values were quantitatively determined via densitometry with ImageJ software.

Cells were rinsed two times with ice-cold PBS and lysed in a lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, and 1% Nonidet P-40) containing protease inhibitors (Roche) and phosphatase inhibitors (1 mM NaVO₃ and 1mM NaF). Forty to seventy micrograms of cell lysate protein were then separated with SDS-PAGE and transferred to a PVDF membrane (Amersham Biosciences). The membrane was blocked with TBST (100 mM Tris-HCl at pH 8.0, 150 mM NaCl, 0.05% Tween-20) containing 5% non-fat dried milk and then incubated with various primary antibodies for 1 hour. After three washes with TBST, the membrane was incubated with a HRP-linked anti-rabbit or anti-mouse secondary antibody (Santa Cruz) for 1 hour and washed again. Bound complexes were visualized using chemiluminescence procedures (NEN Life Science Products) and autoradiography. The primary antibodies used include anti-ERα mAb (Lab Vision), anti-ras-V12 mAb (Oncogene), anti-p53 mAb (Santa Cruz), anti-phospho-Erk1/2 polyclonal Ab (Cell Signaling), anti-phospho-Rb polyclonal Ab and anti- total-Rb polyclonal Ab (Cell Signaling), and anti-GAPDH mAb (Ambion). The density of representative images was quantified using Photoshop software. Phosphorylated proteins was normalized by their total proteins or by GAPDH.