Polyclonal anti p53

Samples were analyzed by western blotting, as described previously (20 (link)). Whole-cell lysates were prepared by lysing cell pellets in a NETN lysis buffer [0.5% Nonidet P-40, 1 mM EDTA, 50 mM Tris (pH 7.4), 12 mM NaCl, 1 mM DTT, 10 mM NaF, 2 mM Na₃VO₄, 1 mM PMSF]. Samples (50 μg) were resolved by SDS-PAGE and transferred to Immobilion-P transfer membranes (Millipore Co., MA USA). The primary antibodies used included monoclonal anti-p16^INK4a, polyclonal anti-Cdk4 and -Cdk6, polyclonal anti-p21, polyclonal anti-p38, monoclonal anti-p-p38, polyclonal anti-p53 and polyclonal anti-pRb (1:1,000, Santa Cruz Biotechnology, Inc., TX, USA). Membranes were washed and incubated with the corresponding HRP-conjugated secondary antibody at 1:10,000. Bound secondary antibody was detected using a chemiluminescence substrate (Advansta, Menlo Park, CA, USA) and visualized on GeneSys imaging system (SynGene Synoptics, Ltd., London, UK).

The following primary antibodies were used: polyclonal anti-SVIP, monoclonal anti-NeuN (Millipore), monoclonal anti-CNS (Millipore), monoclonal anti-GFAP (Beyotime Bio, Shanghai, China), and monoclonal anti-CD11b/integrin am (OX-42). Polyclonal anti-β-actin, polyclonal anti-p53, polyclonal anti-Ki67, polyclonal anti-PSA (A67-B/E3), and anti-AR (N-20) antibodies were obtained from Santa Cruz (TX, USA). Control siRNA and siRNA targeting human SVIP have been previously described [36 ], siRNAs targeting SVIP (5′→3′): siRNA #1, GACAAAAAGAGGCTGCATC; siRNA #2, CCCTTA AGTGCAATGCTAA; siRNA #3, GACAGTTTCATAA AGCATA.