Ecl western blotting detection reagent

Post-blocking the PVDF membrane was incubated with primary anti-Caspase-1 (1:1000, Cat. No.: 106–42020, Adipogen), Caspase-11 (1:1000, Cat. No.: NB120-10454, Novusbio), or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Cat No.: MAB374, Merck Millipore), diluted in 1% (w/v) skim milk in PBST (1xPBS with 0.1% Tween-20) overnight at 4°C with gentle rocking. PVDF membranes were then incubated with species-specific horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 hour at room temperature with gentle rocking. Immunoreactive proteins were detected by applying ECL Western blotting Detection Reagent (Cat No.: 1705060, Bio-Rad) or SuperSignal™ West Femto Maximum Sensitivity Substrate (Cat. No.: 34096, Thermo Fisher Scientific). The results were collected using ChemiDoc™Touch Imaging System (BioRad).

Reagents were purchased from the following sources: rosiglitazone and GW9662 (Sigma, St Louis, MO); RPMI-1640 medium (GIBCO, Invitrogen) and fetal bovine serum (FBS; Shenyang Huibai Biotechnology Co., Ltd.); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT; Biosharp); and ECL Western Blotting Detection Reagent (Bio-Rad, Hercules, CA, USA). Antibodies were selected using polyclonal antibody, PPARγ (Santa Cruz, CA, USA), p-PPARγ (Proteintech, CA, USA), αENaC (Santa Cruz, USA), AQP2 (Santa Cruz, USA), and β-tubulin (Santa Cruz, USA); FITC Alexa 488-conjugated goat anti-rabbit secondary antibodies were from Santa Cruz Biotechnology (USA); and horseradish peroxidase-conjugated anti-rabbit secondary antibodies were from Proteintech (USA).

Proteins were extracted from transfected cells or NDV-infected cells. After the protein samples were boiled for 5 min in 5% SDS–PAGE sample loading buffer, they were separated by SDS-PAGE and transferred to a PVDF membrane for Western blotting. Briefly, the membrane was blocked with 10% skim milk for 12 h at 4 °C. Then, antibodies against BAX, Bcl2, caspase-3 and GAPDH were applied at different dilutions (BAX: 1:500 dilution; Bcl2: 1:500 dilution; caspase-3: 1:500 dilution; and GAPDH: 1:1000 dilution) to detect the proteins. After a 12-h incubation at 4 °C, the membrane was washed three times with Tris-buffered saline containing Tween-20 (TBST). Subsequently, the membrane was incubated with HRP-conjugated anti-rabbit/mouse IgG (1:3000) for 1 h at 37 °C. After four additional washes with TBST, the immunoreactive protein bands were visualized with an ECL Western blotting detection reagent (Bio-Rad), and the results were analyzed using a Tanon-410 automated gel imaging system.

One half of whole frozen retinas were homogenized by passing through a 26-Gauge needle several times in RIPA buffer [1% (v/v) NP-40, 0.1% SDS, 1% (w/v) sodium deoxycholate, 50 mM sodium chloride, 25 mM Tris-HCl pH 8.0] containing an inhibitor protease cocktail. The concentration of proteins in the supernatant was quantified using the BCA kit (660-nm Protein Assay Reagent, Pierce Biotechnology). The same amount of protein was loaded in each lane of SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA). After migration, proteins were transferred to a nitrocellulose membrane, which was blocked in 5% (w/v) skimmed milk in PBS and probed in primary antibody anti-nitrotyrosine, anti-IL-6, anti-IL-8, anti-TNFα, anti-IL-1β (see Table 1) overnight at 4 °C. The membranes were washed three times in phosphate-buffered saline with Tween® (PBST) and incubated in Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (see Table 1) for 1 h at room temperature. The digital images were developed using ECL Western blotting detection reagent (Bio-Rad Laboratories, Hercules, CA, USA) and a chemiluminescence camera (ChemiDoc™ XRS, Bio-Rad). The protein expressions were quantified by densitometry analysis of Western Blots bands using BIO-1D software. The tests were triplicate.

Western blotting was performed according to standard protocols (Toriyama et al., 2012 (link); Toriyama et al., 2017 (link)). Cells were lysed with RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Non-idet P40 substitute, 0.5% Sodium deoxycholate, 0.1% SDS), then the lysate was transferred to a 10% SDS-PAGE polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF membrane followed by incubation with primary antibodies overnight at 4°C. The details of the antibodies are as follows: anti-IFT88 antibody (Proteintech, 13967-1-AP, 1/1000 dilution), anti-Ki67 clone 8D5 antibody (Cell Signaling Technology, #9449, 1/1000 dilution), anti-PDGFRα clone 16A1 antibody (Abcam, ab96569, 1/100 dilution), anti-β-Actin clone C4 antibody (Santa Cruz Biotechnology, sc-47778, 1/1000 dilution). Bands were visualized with an Amersham Imager 600 (GE Healthcare) after incubation with the ECL Western blotting detection reagent (BioRad, RPN2109) or ECL Prime (BioRad, RPN2232).

Cells were lysed in 1X RIPA buffer (Sigma-Aldrich) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). The protein concentration was determined by Bradford method with BSA (Sigma-Aldrich) as the standard. Equal concentration of protein extract were subjected to 12% SDS-PAGE and transferred to PVDF membrane. Blots were labelled with the primary antibodies, ß-catenin (1:1000, Abcam), and GAPDH (1:2000, Sigma). GAPDH was used as a loading control. And following day, HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was added to the blot for 3 hours at RT. Protein expression was detected using ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA) and membranes were imaged using an imaging system (ImageQuant LAS 4000 mini, GE Healthcare).