Chemidoc xrs detection system

Cells were lysed in a lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton-X 100 and a Halt^TM Protease Inhibitor Cocktail (100x, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured by a Qubit^TM Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Equal amount of proteins was run on 10% Tris-tricine gel and blotted to polyvinylidene fluoride (PVDF) membrane with a Bio-Rad Wet Blotting System (Bio-Rad Hungary, Budapest, Hungary). The HER2 receptor was detected by an ErbB2 (HER2) antibody cocktail (Thermo Fisher Scientific, MA5-14057, produced in mouse, 1:500) and an anti-mouse-horseradish peroxidase (HRP) secondary antibody (Thermo Fisher Scientific, 32430, produced in goat, 1:500). As a loading control, β-actin was detected by an anti-actin antibody (Santa Cruz Biotechnology, Dallas, TA, USA, sc-1616, produced in goat, 1:2500) and an anti-goat-HRP secondary antibody (Santa Cruz Biotechnology, sc-2354, produced in mouse, 1:2000). After the addition of enhanced chemiluminescence (ECL) substrate (SuperSignal^TM West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific), the chemiluminescent signal was detected by ChemiDoc XRS+ Detection System (Bio-Rad Hungary).

Whole-cell lysates from BPH-1 cells were prepared in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein were electrophoresed on a mini-PROTEAN TGX precast gel (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After incubation with antibodies, proteins were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, Waltham, MA, USA) and a ChemiDOC XRS⁺ detection system (Bio-Rad, Hercules, CA, USA).

Livers (0.1 g) were homogenized in 0.1 mg/ml phenylmethylsulfonyl fluoride-containing ice-cold RIPA buffer (1 ml) and then the lysates were clarified by centrifugation (12,000 g, 4°C, 15 min), after that the supernatant was collected and stored at −80°C. The samples were denatured with loading buffer (99°C, 10 min), then the equal protein were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with primary antibody according to the dilution ratio provided by manufacturers overnight at 4°C, and then incubated with secondary antibody according to the dilution ratio provided by manufacturers at room temperature for 60 min. The immunoreactivity was detected using the ChemiDoc XRS + detection system (ECL, Bio-Rad, United States). The densitometric analysis was performed with Quantity One^® Image Analyzer software program (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization.

Cells cultured in 10-cm dishes were washed thrice with PBS and then lysed in Radio-Immune Precipitation Assay (RIPA) lysis buffer containing 1% PMSF on ice for 30 min. Following centrifugation (15,000× g, 10 min, 4 °C), the supernatants were incubated at 95 °C for 10 min in 5×SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) loading buffer. After protein separation through electrophoresis on a 15% SDS-PAGE gel, proteins in the gel were transferred to a Polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using 5% nonfat dried milk in TBS-T (10 mM Tris-HCl, pH7.8, 150 mM NaCl and 0.05% Tween-20 (v/v)) for at least 2 h at room temperature. After a short wash with the TBS-T, the membrane was incubated with a primary antibody diluted in the TBS-T by 1000- to 5000-fold for at least 3 h at room temperature. The membrane was washed thrice with the TBS-T and then was incubated with a secondary antibody diluted in the TBS-T by 2500- to 5000-fold for 1 h at room temperature. Following three washings with the TBS-T, protein signals were detected using ChemiDoc XRS⁺ detection system (ECL, Bio-Rad, Hercules, CA, USA). The Quantity One^® Image Analyzer software program (Bio-Rad) was used for quantitative densitometric analysis.

Immunoblotting was carried out as described previously [76 (link)]. The following antibodies were used: mouse anti-Flag M2 monoclonal antibody (1:1500 dilution; Sigma #F3165), rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200, Santa Cruz, CA, USA) and rabbit anti-β-Tubulin polyclonal antibody (1:2500 dilution; Abcam #ab6046, Cambridge, UK). Depending on the species of the primary antibody, the secondary antibody used was either horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:10,000 dilution; Thermo Scientific #31430) or horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution; Cell Signaling Technology #7074, Danvers, MA, USA). Blots were treated with Immobilon chemiluminescent reagent (EMD Millipore, Burlington, MA, USA) and proteins were visualized either on autoradiography film or with ChemiDoc XRS+ detection system (BioRad). Uncropped western blot images are presented in Figures S2 and S3.