β actin clone c4

Protein extraction and immunoblotting were performed and analyzed as described previously with minor adjustments [25 (link)]. For extraction, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 5 mM EDTA, 0.5% w/v Sodium Deoxycholate, and 1% NP-40) supplemented with protease and phosphatase inhibitors (Roche). Lysates were sonicated on ice using the Soniprep 150 MSE at amplitude 10 for 14 cycles (1 second on/1 second off). Insoluble material was removed by centrifugation (10 minutes, 16000x g, 4°C). Protein concentration was determined using a BCA protein assay kit (Pierce/Thermo Fisher Scientific). Proteins samples were separated by SDS-PAGE and transferred to Nitrocellulose membranes (Protran BA-8). Membranes were blocked (1 hour, 5% non-fat dry milk powder (Campina), ambient temp), and incubated with primary antibodies (overnight, 4°C). Antisera: polyclonal goat anti-COL1A1 (1310–01; Southern Biotech), mouse monoclonal β-Actin (clone C4, 08691001; MP Biomedicals, Santa Ana, CA, USA), mouse monoclonal α-Tubulin (clone B-5-1-2, T6074; Sigma-Aldrich). Secondary antisera: polyclonal rabbit anti-goat (P0449; Dako Cytomation, Glostrup, Denmark), rabbit anti-mouse (P0260; Dako). Signals were detected using enhanced chemoluminescence. X-ray films were scanned and analyzed with Quantity One (Biorad).

Cells were lysed using IP buffer (20 mM Tris HCl [pH 7.5], 200 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with Complete Protease Inhibitor mixture (Roche). Samples were resolved by SDS–PAGE and transferred to polyvinylidene difluoride membranes (Perkin Elmer). Membranes were blocked in 5% non-fat milk and incubated overnight at 4°C with the appropriate primary antibody. Membranes were probed with the following primary antibodies: β-actin (clone C4; MP Biomedicals), GAPDH (6C5) (sc-32233; Santa Cruz), p53 (DO-1) (sc-126; Santa Cruz Biotechnology), DNMT1 (clone 60B1220.1; MAB0079; Abnova), PKR (3072; Cell Signaling Technology), phosphorylated PKR Thr 446 (PA5-37704; Thermo Fischer Scientific), EGFR (4267; Cell Signaling Technology), CCND1 (2978; Cell Signaling Technology), HA-tag (3F10; Roche), and ITGA1 (106267; Abcam).
Images were produced using the ChemiDoc XRS imaging system (Bio-Rad).

Cells and tissues were lysed using RIPA buffer. Equal amounts of protein were separated via SDS-PAGE and transferred to a PVDF membrane using Trans-Blot Turbo System (Bio-rad). The following antibodies were used for detection: C/EBPβ (E299) from Abcam; LIN28B (mouse preferred) from Cell Signaling Technology, β-tubulin (GT114) from GeneTex and β-actin (clone C4) (#691001) from MP Biomedicals. For detection, HRP-conjugated secondary antibodies (Amersham Life Technologies) were used. The signals were visualized by chemiluminescence (ECL, Amersham Life Technologies) using ImageQuant LAS 4000 mini imaging machine (GE Healthcare Bioscience AB) and the supplied software was used for the quantification of the bands.

Cells were lysed using IP buffer [20 mM Tris HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 0.5% NP-40] supplemented with Complete Protease Inhibitor mixture (Roche). Samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Perkin Elmer). Membranes were blocked in 5% non-fat milk and incubated overnight at 4°C with the appropriate primary antibody. Membranes were probed with the following primary antibodies: β-actin (clone C4; MP Biomedicals), p53 DO-1 (Santa Cruz Biotechnology, sc-126), PKR (Cell Signaling Technology, 3072), phosphorylated PKR Thr446 (Thermo Fischer Scientific, PA5-37704), p73 (Abcam, ab215038), HA-tag (Roche, 3F10), and Lamin B1 (GeneTex, GTX103292).
Images were produced using the Vilber Fusion FX (Vilber) and the ChemiDoc XRS imaging system (Bio-Rad).