Ecl prime substrate

Control and treated cells, at 70–80% confluency, were harvested on indicated time points with trypsin-ethylenediaminetetraacetic acid (EDTA; Wako, Tokyo, Japan). The cell pellets were lysed further in RIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) and quantified. Ten micrograms of protein (each sample) was resolved in sodium dodecyl sulfate (SDS)–polyacrylamide gel (PAGE) and then electroblotted at methanol-activated polyvinylidene difluoride (PVDF) membrane (Millipore) using a semidry transfer unit (ATTO, Tokyo, Japan). Immunoblotting was performed with indicated antibodies (Supplementary Table 1). PVDF membranes were probed for primary and secondary (HRP-tagged; Santa Cruz) antibodies as described earlier (Singh et al., 2014 (link)). Chemiluminescence detection was performed using enhanced chemiluminescence (ECL) prime substrate (GE Healthcare, Chicago, IL, USA). Densitometric analysis was performed with ImageJ (NIH, Bethesda, MD, USA), and quantitation of each protein in control and stressed cells was normalized with their respective β-actin level.

RIPA lysis buffer supplemented with protease and phosphatase inhibitors was used for the preparation of cellular extracts for WB analysis. Samples were separated by SDS-PAGE and were blotted onto a polyvinylidene difluoride (PVDF-P) WB membrane (Millipore, Billerica, MA, USA) at 60 to 70 V for 1 h. Membranes were incubated with 1% blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany) for 1 h. After three cycles of washing with 0.5% TBS-T buffer (pH7), the membranes were incubated with a peroxidase-conjugated secondary reagent diluted in TBS buffer containing 0.5% blocking reagent for 45 min. After being washed three times with TBS-T buffer, the membranes were incubated for 1 min with ECL Prime substrate (GE Healthcare, Chicago, IL, USA), and signals were detected by Transilluminator Alliance 4.7 (Uvitec Ltd., Cambridge, UK).
Western blot signals were quantified by using ImageJ software according to published protocols [57 (link)], and signals were normalized to associated actin control. Briefly, the normalization factor for every lane was calculated as a ratio between the observed actin signal for every lane and the highest observed signal of the actin for the blot. Normalized experimental signals were calculated by dividing the observed experimental signal by the lane normalization factor.

Cellular extracts for WB analysis were prepared in RIPA lysis buffer supplemented with protease and phosphatase inhibitors, separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride (PVDF-P) WB membrane (Millipore) at 60 to 70 V for 1 h. Membranes were incubated with 1% blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany) for 1 h, followed by 1-h to overnight incubation with primary Abs, three cycles of washing (TBS with 0.05% Tween 20 [TBS-T buffer]), and a 45-min incubation with peroxidase-conjugated secondary reagent diluted in TBS buffer containing 0.5% blocking reagent. After being washed three times with TBS-T buffer (pH 7.5), membranes were incubated for 1 min with ECL Prime substrate (GE Healthcare) and enveloped into plastic wrap. Signals were detected by Transilluminator Alliance 4.7 (Uvitec Ltd., Cambridge, United Kingdom).

Cells (1×10⁶) were seeded on 100 mm cell culture dishes, trypsinized and washed by ice cold PBS twice and lysed on ice with RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors. Proteins were separated by Tris-glycine SDS-PAGE (12% gel) and transferred to nitrocellulose membranes (GE Healthcare). The membrane was blocked in TBST (50 mM Tris-HCl, 150 mM NaCl with 0.01% of Tween-20) containing 5% of non-fat milk for 2 h and consequently incubated with biotinylated antibodies to TRAIL receptors and with streptavidin-HRP (Sigma) for 1 h. ECL Prime substrate (GE Healthcare) and Versa Doc MP4000 documentation system (Bio Rad) were used for visualization of TRAIL receptors. The intensity of protein bands was calculated using the ImageJ software (http://rsbweb.nih.gov/ij/, NIH, Bethesda, Maryland) by option “gel analyzer” tools.

FL-BP180 was expressed in COS7 cells transfected with human BP180 cDNA (35 (link)) and prepared as described previously (36 (link)). Fifty ng of each recombinant human glutathione-S-transferase (GST)-BP180-fusion protein expressed in E. coli spanning most of the BP180 polypeptide were used as an antigen. Immunoblotting and preparation of GST-BP180 fusion proteins were performed as described previously (30 (link)). Serum samples were diluted to 1:100 in 5% non-fat milk-TBS-0.1% Tween-20 and 1:50 000 peroxidase-conjugated anti-human IgG (Sigma-Aldrich, St. Louis, MO, USA) was used as a secondary antibody. Anti-GST (1:3000, Thermo Fisher Scientific, Rockford, IL, USA) with peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich) were used to detect fusion proteins. Protein bands were visualized with ECL Prime substrate (GE Healthcare, Buckinghamshire, UK) on a LAS Imager 3000 (Fujifilm, Tokyo, Japan). Epitope mapping data of 14 age and sex-matched healthy controls from our previous work (30 (link)) were used as a control.