Ecl plus reagent

The HUVECs on Flat, HP1, HP2, and HP3 GHS for 2 days were washed twice with PBS and disrupted with 1 × cell lysis buffer (9803, Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma). A quantitative analysis of the samples was performed using the Bradford assay dye reagent (500–0006, Bio-Rad). The sample protein (10 μg) was boiled in 1 × loading dye for 5 min and subjected to electrophoresis in a 10% polyacrylamide gel with sodium dodecyl sulfate. After transfer to a polyvinylidene fluoride membrane (ISEQ. 00010, Millipore), the membranes were blocked with 5% bovine serum albumin containing 1 × TBST (a mixture of Tris-buffered saline and Tween 20; WH400028806, 3 M) at RT for 1 h. The membranes were incubated with anti-ROCK1 (1:1000; ab45171, Abcam), anti ROCK2 (1:1000; ab71598, Abcam), and anti-GAPDH (1:2000; G8795, Sigma) antibodies at RT for 2 h. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz) in TBST at RT for 1 h. Chemiluminescence was visualized with the ECL Plus reagent (32132, Thermo Fisher Scientific) and recorded on X-ray film.

Cell lysates (10 μg) or purified podoplanin (0.1 μg) were boiled in SDS sample buffer (Nacalai Tesque, Inc., Kyoto, Japan)26 (link). The proteins were electrophoresed on 5–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd.) and were transferred onto a PVDF membrane (EMD Millipore Corp., Billerica, MA). After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the membrane was incubated with primary antibodies or biotinylated lectin (1 μg/ml; Vector Laboratories Inc., Peterborough, UK), then with peroxidase-conjugated secondary antibodies (Dako; 1/1,000 diluted) or streptavidin-HRP (Dako; 1/1,000 diluted), and developed with the ECL-plus reagent (Thermo Fisher Scientific Inc.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Total proteins were extracted by RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China). The protein concentration was measured by the BCA Protein Assay Kit (Thermo Fisher, Waltham, USA). Then, equal amounts of protein were loaded on 8–12% SDS-PAGE gels and transferred onto a PVDF transfer membrane (Millipore, Massachusetts, USA). After blocking with 5% skimmed milk solution, the membranes were incubated with specific primary antibodies overnight at 4°C followed by further incubation with the secondary antibody at room temperature for 2 h. Both primary and secondary antibodies were purchased from Thermo Fisher (Waltham, USA), including anti-β-actin, anti-Nrf2, anti-HO-1, anti-p-NF-κB, anti-IL-1β, anti-TNF-α, anti-Bip, anti-calpain-2, anti-active caspase-12, anti-active caspase-3, anti-Bax, anti-Bcl-2, anti-ACAN, anti-Col-II, anti-Col-I, anti-MMP-13, anti-aggrecanase 1, and secondary antibody IgG. Finally, the protein bands were detected using the ECL Plus Reagent (Thermo Fisher, Waltham, USA), and their densitometry was analyzed with the ImageJ software version 8.0(Bio-Rad, Hercules, USA). With β-actin as control, the expression of total protein was normalized.

The cells were collected for immunoblotting analysis as previously described^{38 (link)}. The primary antibodies were as follows: anti-HIF1α (Novus Biologicals, Littleton, CO); anti-HIF2α (Novus Biologicals, Littleton, CO); anti-GLUT1 (Abcam plc, Cambridge, UK); anti-C/EBPβ (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-α-tubulin (Cell Signaling Technology, Danvers, MA). HRP-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Hercules CA) or anti-mouse IgG (Bio-Rad Laboratories) antibodies were used as secondary antibodies. The signals were detected with the ECL Plus reagent (Thermo Fischer Scientific, Waltham, MA) using the chemiluminescence protocol. For detecting the signals of GLUT1 and HIF-2α, SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific) was used.

The thalamus and kidney from BALB/c mice, as well as ANO2-EGFP-transfected and untransfected NIH/3T3 cells, were lysed in RIPA buffer (Tris-base, 6.5 mM; NaCl, 15 mM; EDTA, 1 mM; NP-40, 1%; and Na-deoxycholate, 0.25%) with 1 mM phenylmethyl sulphonyl fluoride, Na₃VO₄, NaF and protease cocktail. Lysates were centrifuged at 13,000 r.p.m. for 10 min at 4 °C, and the supernatants were isolated for SDS–PAGE. From each sample, 50 μg of protein was resolved on 8% polyacrylamide gels and subsequently electroblotted to polyvinylidene fluoride (Bio-Rad, USA) membranes. Blots were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibodies. Signals were visualized using ECL Plus reagent (Thermo Scientific, USA). The following antibodies were used: rabbit anti-ANO1 (ab53212, 1:500, Abcam, UK), rabbit anti-ANO2 (1:500 (ref. 35 (link))), mouse anti-β-actin (sc-47778, 1:2,000, Santa Cruz, USA), goat anti-rabbit-HRP-conjugated (31460, 1:5,000, Pierce, USA) and goat anti-mouse-HRP-conjugated (31430, 1:5,000, Pierce).

5 × 10⁶ cells at the logarithmic growth stage were collected. To extract the total proteins, RAPI lysate (Beyotime, Nanjing, China) containing protease and phosphatase inhibitors was used. The relevant protein concentration was measured using a BCA kit (Thermo Fisher Scientific, MA, USA). After quantification, the proteins were separated using SDS-PAGE. These proteins were transferred to polyvinylidene fluoride (PVDF) membranes and sealed in 5% degrease milk for 1 h. The blots were incubated at 4 ℃ overnight with the primary antibodies against ASC (CST, 67,824, 1:1000), pro-caspase-1 (Abcam, ab32499, 1:10,000), caspase-1 (Santa, sc-56036, 1:500), IL-1β (CST, 12,703, 1:1000), IL-18 (CST, 54,943, 1:1000), and GAPDH (Abcam, ab181602, 1:10,000). The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated antibodies at 23 °C for 1 h and then developed using ECL-Plus reagent (Thermo Fisher Scientific). We observed the membranes using gel imaging system and analyzed the findings.