Phosphatase inhibitor cocktail

The MAPK assays were described previously with some modifications (16 (link)). Briefly, rice suspension cells were subcultured in fresh medium for 3 days. Aliquots of 500 mg of cells were suspended in 5 mL preincubation medium (3% sucrose and 10 mM MES [morpholineethanesulfonic acid] in 5% culture medium [pH 5.8]) and incubated under culture conditions for 4 h. Total proteins were extracted with plant radioimmunoprecipitation assay (RIPA) lysis buffer (P0045; Beyotime Biotechnology, Shanghai, China) and phosphatase inhibitor cocktail (B15001; Bimake, Shanghai, China) from rice suspension cells subjected to treatment with ShAM1 or ShAM1-digested cell wall extract. Analysis was carried out by SDS–PAGE and Western blotting using a phosphorylation-specific p38 MAPK antibody (1:5,000 dilution) (Cell Signaling).

CD8⁺ and Vγ9Vδ2⁺ T cells were treated with 1α,25(OH)₂D₃ (50 nM) three times at 1-day intervals in vitro. T cells were stimulated with anti-human CD3 (5 µg/mL) and anti-human CD28 (1 µg/mL) at the indicated time points (0 s, 30 s, 60 s, 3 min, 5 min, and 10 min). Subsequently, cells were harvested and lysed with RIPA buffer (Beyotime, P0013K), which included phosphatase inhibitor cocktail (Bimake, B15001). Total proteins (20–40 µg) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and electrotransferred to 0.22 µm PVDF membrane with the PowerPac wet-blot system (Bio-Rad). Approximately 5% BSA was used to block membranes for 1 hour, and then incubated with the indicated primary antibodies overnight at 4℃. Primary antibodies included p-Zap70 (CST, 2717), Zap70 (CST, 2705), LAT (9166), p-LAT (CST, 3584), PLCγ1 (CST, 2822), p-PLCγ1 (14008), VDR (CST, 12550), PD-1 (CST, 84651), and β-actin (CST, 3700). After six times washed in TBST for 1 hour, the membranes were incubated with HRP (Horseradish Peroxidase)-linked secondary antibody at a dilution of 1:2000 (CST, 7074) at room temperature for 2.5 hours. Finally, membranes were washed five times in TBST, and then detected by Bio-Rad ChemiDoc MP Gel imaging system (Bio-Rad).

Total proteins of kidney tissues or TCMK-1 cells were extracted using radio-immune precipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Bimake, Houston, TX, United States). The protein concentrations were determined using the BCA protein assay kit (Biosharp, Hefei, China). Equal amounts of protein lysate were separated by 10–12% SDS-polyacrylamide gel in Tris/SDS buffer and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). The membranes were blocked in 5% non-fat milk (w/v) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h at room temperature and incubated with corresponding primary antibodies at 4°C overnight. After washing, the membranes were further incubated with HRP-conjugated secondary antibodies (1:10,000) at room temperature for 1 h. The immunoreactive bands were evaluated to visualize the expression of designated proteins using the chemiluminescence detection system through the peroxidase reaction, and the images of the bands were recorded with the Chemi Doc MP imaging system (Bio-Rad, United States). GAPDH was used as the internal loading control. The films were analyzed with ImageJ software (National Institute of Health, Bethesda, MD, United States). All experiments were repeated at least three times.

Cells were lysed by incubation in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl (pH 8.0) supplemented with Protease Inhibitor Cocktail (Bimake) and Phosphatase Inhibitor Cocktail (Bimake) for 30 min on ice. Next, the cell extracts were sonicated with a VirSonic 100 ultrasonic cell disrupter and stored at −70°C. The protein concentration was measured by the Bradford assay. Aliquots of each sample were separated by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham/GE Healthcare). The membranes were blocked for 1 h in 2% ECL Advance blocking reagent (GE Healthcare) or 2% bovine serum albumin (BSA) (Sigma) in PBS containing 0.1% Tween 20 (PBS-T) followed by incubation overnight at 4°C with a primary antibody diluted in PBS-T containing 2% blocking reagent or 2% BSA. After three washes with PBS-T, the membranes were incubated for 1 h with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG) in PBS-T containing 2% blocking agent or 2% BSA. The immunoblots were visualized using a Pierce ECL plus western blotting substrate.