Amersham ecl prime detection reagent kit

The protein extracts were dissolved in RIPA buffer for 30 min on ice, and then the samples were centrifuged at 12,000 × g for 15 min to collect the supernatant containing soluble proteins. The protein concentration was measured using the BCA Assay (Bio-Rad, Hercules, CA, USA). The protein solution was mixed with 4×loading buffer and heated at 100 °C for 10 min before being subjected to WB. The total protein samples (~ 30 μg) were subjected to SDS‒PAGE and then blotted onto 0.2 μM polyvinylidene fluoride (PVDF) membranes (#1620177; Bio-Rad). The membranes were blocked with 0.05% Tween 20 (#P9416; Sigma Aldrich) v:v in Tris-buffered saline (TBS) (TBST) (#ET220; Euromedex) supplemented with 5% nonfat powdered milk (w:v in TBS), followed by overnight incubation at 4 °C with primary antibodies specific for KLF5 (#AF3758; 1:1000; R&D Systems), COX2 (#66351-1-Ig; 1:1000; Proteintech), CyclinD1 (#55506; 1:1000; Cell Signaling Technology) and Vinculin (#13901; 1:2000; Cell Signaling Technology). The membranes were washed with TBST three times for 10 min before incubation with HRP-conjugated secondary antibody for 1 h at room temperature. Next, the membranes were washed again and subjected to chemiluminescence detection using the Amersham ECL Prime detection reagent kit (GE Healthcare, Piscataway, NJ, USA) on an ImageQuant LAS 4000 software-assisted imager.

Immunoblotting was performed according to standard procedures of the NuPAGE® electrophoresis system (Invitrogen). In brief, protein extracts were obtained by lysing cells in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors cocktail, which were separated on 4–12% NuPAGE® Novex® Bis-Tris gels in the NuPAGE® MES SDS Running Buffer and electrotransferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) in the Tris-Glycine (TG) buffer. Membranes were incubated for 1 h in 5% non-fat powdered milk dissolved in TBST (Tris-buffered saline containing 0.05% Tween 20) to saturate unspecific binding sites, followed by an overnight incubation (4 ^oC) with primary antibodies specific for p-EIF2α, ab32157, EIF2α, ab5369, ATF4, ab31390, XBP1, ab37152, β-Actin, ab20272 (Abcam, Cambridge, UK); Hexokinase II, 2867 (Cell Signaling Technology, Danvers, USA). Membranes were washed 5× times with TBST and revealed with suitable horseradish peroxidase-conjugated secondary antibodies (Southern Biotechnologies, Birmingham, AL, USA) for another 2 h at room temperature, followed by additional washing steps and chemiluminescence-based detection with the Amersham ECL Prime detection reagent kit and the ImageQuant LAS 4000 software-assisted imager (GE Healthcare, Piscataway, NJ, USA).

Samples were mixed with 4× Pierce™ LDS Sample Buffer (#84788, ThermoFisher Scientific, Waltham, MA, USA) including DTT (0.3M) and denatured for 5 min at 75 °C. For Western blot analysis, 5 μL (fractions 6–8 in UF+SEC) or 25 μL (other samples) of protein sample was separated using a 4–20% SDS-PAGE (Mini-PROTEAN^® TGX Stain-Free™ Protein gels, #4568094, BIO-RAD, Hercules, CA, USA) and MOPS running buffer and transferred to nitrocellulose membranes using a BIORAD Trans-Blot SD semi dry transfer system. Membranes were blocked for 45 min (PBS, 0.1% Tween 20, 5% dried milk) and primary antibodies against Tsg101 (ab83, Abcam, Cambridge, UK), Alix (ab117600, Abcam, Cambridge, UK), Tamm-Horsfall protein (uromodulin, ab733, Millipore, Burlington, MA, USA) and CD9 (#MA1-80307, Invitrogen, Carlsbad, CA, USA) were used at 1:1000 dilution o/n at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit anti-goat (#P0160, Dako, Agilent Technologies, Santa Clara, CA, USA, 1:5000) and rabbit anti-mouse (#P0260, Dako, 1:5000)) were used and chemiluminescence activated using the Amersham ECL Prime detection reagent kit (#RPN2232 GE Healthcare, Chicago, IL, USA). Bands were visualised using the signal accumulation mode with a Bio-Rad ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA).