Luminata forte ecl detection system

Cells were washed with phosphate-buffered saline (PBS) containing 20 mM N-ethylmaleimide (NEM) for 1 min and then lysed with 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–buffered saline, pH 6.8, supplemented with 20 mM NEM and protease inhibitors for 20 min on ice. Postnuclear supernatants (PNSs) were collected by centrifugation at 10,000 × g for 10 min. Samples were denatured and reduced in dithiothreitol (DTT)-containing sample buffer for 10 min at 65°C and separated by SDS–PAGE. Proteins were transferred to PVDF membranes with the Trans-Blot Turbo Transfer System (Bio-Rad, Cressier, Switzerland). Membranes were blocked with 10% (wt/vol) nonfat dry milk (Bio-Rad) and stained with the aforementioned primary antibodies and horseradish peroxidase–conjugated secondary antibodies. Membranes were developed using the Luminata Forte ECL detection system (Millipore, Schaffhausen, Switzerland), signals were detected with the ImageQuant LAS 4000 system in the standard acquisition mode (GE Healthcare Life Sciences, Glattbrugg, Switzerland), and bands were quantified using the Multi Gauge Analysis tool (Fujifilm). For each antigen, the linearity of the detected signal range was ensured with appropriate loading controls.

After the respective treatments, HEK293 cells were washed with ice-cold phosphate-buffered saline (PBS) containing 20 mM N-ethylmaleimide (NEM) then lysed with 2% CHAPS (in HEPES-buffered saline [HBS], pH 7.4) or RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate in HBS, pH 7.4) supplemented with 20 mM NEM and protease inhibitors. PNS and lysis buffer-insoluble pellet were collected after centrifugation at 4°C 10,600 × g for 10 min. PNS was denatured for 5 min at 95°C with the addition of 100 mM dithiothreitol and subjected to SDS–PAGE. Following in-gel TMR imaging with Typhoon FLA 9500 (Software Version 1.0) with a 532-nm laser, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 8% (wt/vol) nonfat dry milk (Bio-Rad) in Tris-buffered saline (TBS)-T and stained with primary antibodies diluted in TBS-T followed by HRP–conjugated secondary antibodies or HRP–conjugated Protein A diluted in TBS-T. Membranes were developed using Luminata Forte ECL detection system (Millipore) and signals were captured with FusionFX chemiluminescence imaging system (VILBER). TMR bands were quantified using the ImageQuant TL software (Molecular Dynamics, GE Healthcare).