Fusion fx chemiluminescence imaging system

Following 72 h of transfection, MCF-7 cell lysates were prepared using ProteoJET™ mammalian cell lysis reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The protein concentration of the cell lysate was determined using Bradford assay reagent (cat. no. 5000006, Bio-Rad Laboratories, Inc.). Protein samples (30 µg) were loaded into the well of 12% SDS-PAGE. Following separation with 12% SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with 5% (w/v) skimmed milk in TBST buffer for 1 h at room temperature prior to detection with rabbit primary β-actin (dilution, 1:5,000; cat. no. ab8227; Abcam) and Chka (dilution, 1:1,000; cat. no. ab88053; Abcam) antibodies overnight at 4°C. The membrane was washed ≥3 times with TBST (0.1% (v/v) Tween-20 in TBS) buffer prior to incubation with HRP-conjugated secondary goat anti-rabbit IgG antibody (dilution, 1:5,000; cat. no. 12–348; Sigma-Aldrich, Merck KGaA) at room temperature for 1 h. The membrane was then washed three times prior to detection with SuperSignal™ West Femto maximum sensitivity substrate (cat. no. 34094, Thermo Fisher Scientific, Inc.). The signal was visualized using the FUSION FX chemiluminescence imaging system (Vilber Lourmat) and the band intensities were analyzed using ImageJ 1.49b software (National Institutes of Health).

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).