Ecl western blotting detection system

Total cell lysates were prepared using RIPA buffer^{24 (link),26 (link)}. Each protein sample (40–50 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes^{24 (link),26 (link)}. Membranes were incubated with primary antibodies at 4 °C overnight. Antibodies against FHC (B-12) (1:200, sc-376594), p53 (A-1) (1:500, sc-393031), BAX (B-9) (1:500, sc-7480), and Bcl-2 (100) (1:500, sc-509) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, Texas); antibodies against Caspase-9 (1:500, #9502), Caspase-8 (1C12) (1:500, #9746), and Fas (C18C12) (1:500, #4233) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Membranes were then incubated with secondary antibodies, HRP-conjugated goat anti-mouse IgG (1:2000, sc-516102; Santa Cruz Biotechnology, Dallas, Texas) and HRP-conjugated mouse anti-rabbit IgG (1:2000, sc-2357; Santa Cruz Biotechnology, Dallas, Texas), and immunoreactive bands were visualized with the ECL western blotting detection system (Santa Cruz Biotechnology, Dallas, Texas). To ensure equal loading of proteins a goat polyclonal anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396; Santa Cruz Biotechnology) was used. The protein band intensity on western blots was quantified and normalized to that of γ-Tubulin by using ImageJ software (http://rsb.info.nih.gov/ij/).

Total cell lysates were prepared using RIPA buffer^{38 (link),39 (link)}. Each protein sample (40–60 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies against c-Myc (C33, sc-42), Cyclin D2 (B-6, sc-376676), Vimentin (V9, sc-6260), SNAI1 (E-10, sc-393172), SLUG (A-7, sc-166476) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, Texas (Santa Cruz Biotechnology, Dallas, Texas) while antibodies against Fas (4233 S) and Caspase 3 (9662 S) were purchased from Cell Signalling Technology, Leiden, Netherlands). Membranes were then washed and incubated, for 2 h, with secondary antibodies HRP-conjugated goat anti-mouse IgG (sc-2005) and HRP-conjugated goat anti-rabbit IgG (sc-2357) (Santa Cruz Biotechnology, Dallas, Texas), and immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology, Dallas, Texas). To ensure equal loading of proteins was used a goat polyclonal anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology). Experiments were performed three times and representative images are reported.

Total cell lysate was prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS and a mixture of protease inhibitors). Western blot analysis was performed using the same amount of protein. Protein concentration was determined by the Bradford (Sigma-Aldrich, St. Louis, MO, USA) method and equal quantities were subjected to Western blot analysis. SDS-PAGE-separated proteins were electroblotted onto a nitrocellulose membrane. The blots were incubated overnight at 4 °C with the following primary antibodies: anti-ERRα (ab76228; 1:1000); anti-SR-BI (ab52629; 1:1000); anti-HMGCR (ab215365; 1:500); from Abcam, Cambridge, UK; anti-Cyclin D1 (sc-8396; 1:500), anti-PARP-1 (sc-7150; 1:2000) and anti-GAPDH (sc-32233; 1:10,000) from Santa Cruz Biotechnology, Dallas, TX, USA. Anti βactin (ab8226; 1:1000; Abcam) was used as a loading control. All antibodies were incubated with appropriate horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected by the ECL Western blotting detection system (Santa Cruz Biotechnology, sc-2048). All the whole western blot figures can be found in the Supplementary Materials.

Total cell proteins and the cytoplasmic and nuclear fractions were obtained from 70% confluent cell cultures. Western blotting (WB) was performed as previously described [95 (link)]. Blots were incubated with primary antibodies (overnight, 4 °C) and then with appropriate horseradish-peroxidase-conjugated secondary antibodies (1 h, room temperature). Immunoreactive bands were detected using the ECL Western blotting detection system (Santa Cruz Biotechnology (Dallas, TX, USA), sc-2048). Images were captured using UltraCruz Autoradiography Film (Santa Cruz Biotechnology) or iBright Imaging System (Thermo-Fisher (Waltham, MA, USA). The images from films were acquired using an Epson Perfection scanner (Epson, Japan) using Photoshop software (Adobe) (https://www.adobe.com/au/products/photoshop.html, accessed on 20 July 2023). The optical densities of the spots were analyzed by using ImageJ software (NIH) (https://imagej.en.softonic.com/download, accessed on 20 July 2023).

RIPA lysis buffer was used to lysate cells.³⁴ Western blot analysis was performed using equal quantity of protein. Protein concentration was determined by Bradford method (Sigma‐Aldrich) and equal amounts were subjected to Western blot analysis. All membranes were incubated for 12 hours at 4°C with antibodies against sirt1, ERα, IGF1R, CCND1, vimentin, N‐cadherin, bax, bcl‐2, parp1 and cytochrome c (all from Santa Cruz Biotechnology, Santa Cruz CA, USA). Membranes were incubated with HRP‐conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA), and immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology, Santa Cruz CA, USA). GAPDH (Santa Cruz Biotechnology, Santa Cruz CA, USA) or VDAC1/porin (Abcam, Cambridge, UK) antibodies were used as a loading control.