β actin clone c4

Proteins were extracted using a buffer composed of: 10 mM PIPES; 300 mM NaCl; 1 mM EDTA; 300 mM sucrose; 1 mM MgCl₂; 0.5% TritonX-100; cOmplete™, EDTA-free Protease Inhibitor Cocktail 1x (Roche, Mannheim, Germany); DTT 1 mM; NaF 1 mM; Na₃VO₄ 0.2 mM. Protein quantification was done using Protein Assay Dye Reagent Concentrate (Bio-Rad, Munich, Germany). The Western blotting analysis was performed using 40 µg protein and incubated with the following antibodies: HPV16 E6 (2 µg/mL, #849 Arbor Vita Corporation); HPV16 E7 NM2 (kindly provided by Prof. Martin Müller, DKFZ); p53 (DO-1, Santa Cruz-126); pRb (C-15, Santa Cruz-50); β-Actin clone C4 (Millipore 691001). Intensities of bands were quantified by ImageJ.

Cells were lysed using Mammalian Protein Extraction Reagent (M-PER) (Thermo Fisher Scientific) containing protease inhibitor cocktail (cOmplete tablet; Roche, Mannheim, Germany) and phosphatase inhibitor cocktail (PhosSTOP; Roche). Equal amounts of proteins from cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked using 5% ECL blocking agent (GE Healthcare, Chicago, IL) in Tris-buffered saline containing 0.05% Triton X-100. Proteins of interest were detected using primary antibodies against phosphorylated forms of AKT (Ser473; Cell Signaling Technology, Danvers, MA) and ERK1/2 (PROMEGA, Madison, WI), and against IGFBP-3 (B-5; Santa Cruz Biotechnology, Dallas, TX), AKT (Cell Signaling) and β-actin (clone C4; Millipore, Billerica, MA). Following incubation with primary antibodies, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Detected proteins were visualized with ECL Western Blotting Detection reagents (GE Healthcare). Protein expression levels were quantified using ImageJ^{56 (link)}. Active MAPK/ERK and p-AKT expression levels were normalized to β-actin and total AKT protein levels, respectively.

The cell pellets were lysed in lysis buffer (Cell Signaling) with a protease inhibitor cocktail (Sigma Aldrich). Total cell lysates were subjected to western blotting with antibodies against ICP0 (clone 5H7; Santa Cruz), ICP4 (clone H943; Santa Cruz), ICP8 (clone 10A3; Santa Cruz), TRIM28 with phosphorylated Ser473 (clone 11G10SC; BioLegend), TRIM28 with phosphorylated Ser824 (Abcam), TRIM28 (Sigma-Aldrich), ILK (Genetex), Akt with phosphorylated Ser473 (Genetex), Akt (Genetex), FLAG (clone M2; Merck), SUV39H1 (clone MG44; mouse ascites; Millipore), and β-actin (clone C4; Millipore) and HRP-conjugated secondary antibodies. Protein bands were developed by an enhanced chemiluminescence substrate kit (Millipore) and detected by UVP system. The intensity of the protein bands was measured by ImageJ software.