β actin monoclonal antibody

Protein extraction, quantification, and western blot was performed as previously described (19 (link)). The membranes were probed with SLC12A6 (Boster Bio, Pleasanton, CA), MMP-3 (Boster Bio), SSX2IP (Novus Biologicals, Centennial, CO), TRAIL-R4 (Boster Bio), GBP1 (Novus), GUCY1A3 (Novus) or TRPC4 (Boster Bio) primary antibodies. Membrane developing was conducted by enhanced chemiluminescence and autoradiography in an SRX-101A film processor (Konika Minolta, Japan). The ImageLab software was used to quantify the density of bands. All membranes were reprobed with a β-actin monoclonal antibody (Sigma) as a loading control.

Culture cells were lysed in lysis buffer (Roche, IN) and centrifuged at 12,000g for 10 min to collect soluble proteins. Tissues were homogenized on ice in 300µl of 50mM Tris.Cl buffer (pH 7.0), followed by centrifugation at 12,000g for 10 min to collect supernatants. For Western blot, proteins (30 ~ 50 µg) were separated on 12% SDS-PAGE and blotted onto a pure nitrocellulose membrane (Bio-Rad, CA) at 180 mA for 2 hours. After being blocked with 5% skim milk in PBS at room temperature for 45 min, membranes were incubated with AKR1B10 (1:500) or acrolein (1: 500, Advanced Targeting System, CA) antibodies at 4^oC overnight, followed by incubation with goat anti-rabbit or anti-mouse IgG (1:2000; Sigma, MO) for 1 hour. Antibody binding signals were detected using enhanced chemiluminescence system (Pierce, IL). Protein loading amount was normalized by re-probing the membrane with β-actin monoclonal antibody (1:40,000; Sigma, MO).

Total protein was extracted with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and the concentration was assessed by Coomassie Brilliant Blue R250 staining. After electrophoresis in Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), the protein was transferred to polyvinylidene fluoride (PVDF) membranes and then incubated with anti-calponin 2 monoclonal antibody 1D2, produced at Wayne State University, USA), PCNA polyclonal antibody (Cell Signaling Technology, USA), p-Erk1/2/Erk1/2 antibody (Cell Signaling Technology, USA), p-JNK/JNK antibody (Cell Signaling Technology, USA; Abcam, England), p-p38/p38 antibody (Cell Signaling Technology, USA), p-STAT/STAT antibody (Cell Signaling Technology, USA), p-AKT/AKT antibody (Cell Signaling Technology, USA; Abcam, England), p-p65/p65 NF-κB antibody (Cell Signaling Technology, USA), E-cadherin antibody (Cell Signaling Technology, USA) and β-actin monoclonal antibody (Sigma, USA) as internal control at 4°C overnight. After washes, secondary antibodies conjugated with horseradish peroxidase (Zhong Shan Goldenbridge Biotech, China) was used for chemiluminescence (Bio-rad, CA, USA) detection of the specific proteins. Densitometry quantification of the bands was performed with Quantity One software (Bio-rad, CA, USA).

Cell-surface biotinylation was carried out with the Pierce Cell Surface Protein Isolation Kit (Pierce, Rockford, IL, USA). tsA201 cells were transfected with pRcCMV-hClC-1, T335N, or both constructs (10 μg each). One day after transfection, cell-surface proteins were labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-biotin). In brief, cells were washed with ice-cold PBS twice, and Sulfo-NHS-SS-biotin was added and incubated at 4°C, with constant rotation for 30 min. Excess biotin was quenched with quenching solution. Cells were treated with lysis buffer and centrifuged at 10,000 g for 2 min at 4°C. Clear supernatant was reacted with immobilized NeutrAvidin gel slurry in columns to isolate surface proteins. Columns were washed and protein eluted in sample buffer that contained DTT. Surface proteins were separated on a SDS-PAGE gel, and samples (1 μg) were analyzed by Western blotting using a monoclonal anti–ClC-1 antibody (MyBiosource). Filters were also immunoblotted with β-actin monoclonal antibody (Sigma-Aldrich) as control. Total protein density was standardized as the ratio of input signal to β-actin signal, normalized to WT control. Surface protein density was measured as the ratio of surface signal to cognate total β-actin signal, followed by normalization to WT control.