Ecl prime western blotting detection kit

The cultured HNF1A-inducible cell line was dissociated and similar numbers of cells were pelleted. The cell pellets were washed twice with PBS, and lysed into RIPA buffer (Sigma, #R0278) according to the manufacturer’s instructions. The proteins were mixed with Sample buffer (Wako, # 198-13282), denatured, separated by SDS-PAGE on Mini-PROTEAN TGX Gels (Bio-Rad, #456-1086), and were transferred to Immun-Blot PVDF membranes (Bio-Rad, #162-0177). The membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), washed in TBST, and then incubated with the primary antibodies against HNF1A (Abcam, ab96777; 1:500) and β -ACTIN (Cell Signaling, #4970; 1:1000) in 2% BSA/TBST overnight at 4 °C. The membranes were washed three times in TBST and then incubated with horseradish peroxidase-conjugated anti-rabbit antibodies (GE Healthcare, NA934) (1:2000) for 1 h at RT. The immunoreactivity was detected using an ECL Prime Western Blotting Detection Kit (GE Healthcare, RPN2232) and LAS-4000 (Fujifilm).

Proteins from the lysate of transfected HepG2 cells (60 µg) or liver (50 µg) were blotted and immunostained with anti-human FXI polyclonal antibody (Enzyme Research Laboratories, Swansea, UK) and anti-human β-actin monoclonal antibody (Sigma-Aldrich, Madrid, Spain). Additionally, we collected and lyophilized 500 µL supernatants from HepG2 and PLC/PRF/5 using CentriVap Concentrator (Labconco, Kansas, MO) and 24 µL were blotted and immunostained with anti-human FXI polyclonal antibody. FXI and β-actin were immunodetected with the appropriate secondary antibody labeled with peroxidase (GE Healthcare, Barcelona, Spain). Detection was performed using ECL Prime Western Blotting Detection Kit (GE Healthcare, Barcelona, Spain) and ImageQuant LAS 4000 Imager. Densitometric analysis was performed with ImageJ software (http://rsb.info.nih.gov/ij/). Data were expressed as changes relative to the values of the cells transfected with SCR, taken as 100%.

HaCaT cells were incubated in serum-free DMEM for 24 h and treated with higenamine (5, 10, 20 μM) for 1 h and treated fine dust. For extracellular matrix proteins, the cultured medium was collected. For intracellular proteins, cell lysates were prepared using cell lysis buffer (50 mM tris-HCl (pH 8.0), 0.15 M NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride 1 mM Na₃VO₄). Both the cultured medium and cell lysates were harvested on ice and centrifuged at 13,000 rpm for 10 min, and supernatant was collected. Protein concentrations were measured using a protein assay kit (Bio-Rad, Hercules, CA, USA). The proteins were electrophoretically separated using an 8%, 10% SDS-polyacrylamide gel and transferred onto 0.2 μm PVDF membranes (Little Chalfont, UK). The membrane was blocked in 5 % non-fat milk or 5% bovine serum albumin (BSA) for 1 h. The membrane was incubated with specific primary antibody for 4 °C overnight. After washing the membrane, the HRP-conjugated secondary antibody (Santa Cruz) was combined on membrane for room temperature 1 h. Protein bands were detected using an ECL prime Western blotting detection kit (GE Healthcare, Chicago, IL, USA). The Western blot data were analyzed using quantitative analysis in Image Studio software 5.5.4 (LI-COR, Lincoln, NE, USA).

After a 4-week incubation of fibroblasts with carbamylating agents, cells were washed with PBS and lyzed using a specific buffer (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 1% Igepal CA-630; 0.5% (m/v) sodium deoxycholate; 0.1% sodium dodecyl sulphate) containing 1% (m/v) protease inhibitors cocktail (Hall Protease Inhibitor Cocktail, Thermo Scientific, Rockford, IL, USA). Protein content in cell lysates was determined using Bradford assay (Protein Assay Dye Reagent Concentrate, Bio-Rad, Hercules, CA, USA). Ten micrograms of proteins were then separated by 8% (m/v) SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with Tris-buffered saline (Tris HCl 20mM pH 7.5, NaCl 150mM) containing 0.1% Tween (v/v) (TBS-T) and 5% (w/v) fat-free dehydrated milk, at room temperature for 1 hour. After three washes with TBS-T, the membrane was incubated overnight at 4°C with an anti-ubiquitin antibody directly coupled to horseradish peroxidase (Santa Cruz, Dallas, USA, used at 1/120 dilution in TBS-T containing 1% (m/v) dry milk). Finally, detection was performed using an ECL Prime Western Blotting Detection kit (GE Healthcare) according to the manufacturer’s protocol.