Rabbit anti pcna 13110

Cells were lysed in Tris-HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM and protease inhibitors. For phospho p65, 2 mM Na orthovanadate and 5 mM Na butyrate were also added. Lysates were separated on 8% or 10% acrylamide gel and immunoblotted using standard procedures. Rabbit anti-Hsp70, sc-33575 (Santa Cruz Biotechnology, CA), rabbit anti-PCNA, #13110 (Cell Signaling Technology Inc., Danvers, MA); mouse anti-b3-tubulin (TU-20), #4466 (Cell Signaling Technology Inc, Danvers, MA); mouse anti-GFAP, MAB360 (Merck Millipore, Darmstadt), rabbit anti-p65, #3034S (Cell Signaling Technology Inc, Danvers, MA); rabbit anti-phospho p65 (P-p65), #3033S (Cell Signaling Technology Inc, Danvers, MA); mouse anti-GAPDH, ab8245 (AbCam, Cambridge, UK) and HRP-conjugated secondary antisera (Santa Cruz Biotechnology, CA) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK) and images were acquired using the BioRad ChemiDoc MP Imaging System (BioRad, Hercules, CA). Densitometric analysis was performed using the BioRad associated Image Lab Software (BioRad, Hercules, CA). Values are expressed as fold over internal control, represented by GAPDH, that does not change significantly in the proteome profiles.