Rabbit anti p53

The following primary antibodies were used for immunohistochemical analysis: rabbit anti-RIG-G (a gift from Dr. Jianhua Tong), rabbit anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Ki-67, mouse anti E-cadherin, and rabbit anti-vimentin (All three antibodies from Cell signaling technology, Boston, USA). The lungs were infused with 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) and incubated for 24 h. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. For immunohistochemical analysis, sections were deparaffinized and boiled for 15 min in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Unspecific tissue peroxidases were blocked by 3% (v/v) H₂O₂. Sections were incubated with primary antibodies in 4℃ overnight. Secondary antibody incubation and staining were performed using the EnVision®+ System–HRP (DAB) kit (Dako, Carpinteria, CA, USA), according to the manufacturer’s recommendations. The sections were counterstained with hematoxylin and mounted.

The presence of various proteins in the samples was confirmed by 10 % SDS-PAGE, followed by electroblotting to PVDF membranes and detection using the respective primary antibodies: rabbit anti-p53 (SantaCruz, sc-6243, 1:1,500), rabbit anti-15-phosphoserine (SantaCruz, sc-101762, 1:2000), rabbit anti-Mekk1 (SantaCruz, sc-252, 1:1,500), mouse anti-β–tubulin (SantaCruz, sc-5286, 1:1,500), and rabbit anti-PARP-1 (SantaCruz, sc-74469, 1:1,500), mouse anti-β-actin (SantaCruz,, 1:10,000), and mouse anti-Mdm2 (SantaCruz, sc-965 1:2000). Alkaline phosphate-conjugated secondary anti-rabbit (Sigma, A8025) or anti-mouse (Sigma, A7434) antibodies were used at 1:10,000 dilution. Following equilibration in chemiluminescence buffer (0.1 M diethanolamine, 1 mM MgCl₂, pH 9.5) for 5 min, the membranes were incubated with substrate CDP-Star (Amersham) for 5 min and exposed to film (RPI). For re-probing, the blots were stripped of primary and secondary antibodies using 20 mM Tris-HCl, pH 6.8 containing 1% SDS and 100 mM 2-mercaptoethanol at 55°C for 45 min on a rotating wheel (20 rpm), followed by extensive washing with water and blocking with 5% non-fat dry milk for at least 2 h prior to incubating again with primary antibodies.

Cells were lysed in cell lysis buffer (cell signaling technology, Boston, USA) supplemented with protease and phosphatase inhibitors (Roche, Germany). Briefly, 30 μg total protein were loaded on 10% SDS polyacrylamide gels, electrophoresed, and blotted onto Hybond-C Extra membranes (Amersham Bioscience, Buckinghamshire, UK). Primary antibodies used for western blot analysis included the following: rabbit anti Rig-G (a gift from Dr. Jianhua Tong), mouse anti-cyclin D1, rabbit anti-p21, mouse anti-E-cadherin, rabbit anti-vimentin, rabbit anti-p-Akt (Ser473), rabbit anti-p-MDM2(Ser166), rabbit anti-Bcl-2, mouse anti-β-actin (all from Cell Signaling Technology) and rabbit anti-p53(Santa Cruz Biotechnology). HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology) or rabbit anti-mouse (Dako) was used as secondary antibody. Where indicated, cells were treated with pifithrin-α (10μM) prior to protein lysis.

EGCG (>95%) was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-human proliferating cell nuclear antibody (PCNA) (FL-261), rabbit anti-human/rodent p21 antibody, rabbit anti-p53 and goat anti-human actin polyclonal antibody (I-19) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.