Polyvinylidene difluoride pvdf membrane

The extraction of total protein from cells was performed employing RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). After the samples were mixed with 5×loading buffer and denatured in boiling water for 5 min, the separation of the proteins was completed via SDS-PAGE, followed by subsequent transfer of separated proteins onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China). After that, the membranes were blocked in 5% skimmed milk for 2 h at room temperature and then incubated with primary antibodies against MAPK1 (1: 5000; ab257525; Abcam, Shanghai, China) and β-actin (1: 2000; ab5694; Abcam, Shanghai, China) overnight at 4 °C. Next, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:1000, Beyotime, Shanghai, China) at room temperature for 2 h. The development of protein bands was conducted employing an enhanced chemiluminescent kit (Biozym, Hessisch Oldendorf, Germany).

Protein was extracted from tea leaf samples treated with AsODN-CsGGT4 or sODN-CsGGT4. The quantification of protein concentration was conducted using a Bradford protein assay kit (Beyotime, Shanghai, China). A 12.5% SDS-PAGE gel was employed to separate 20 micrograms of plant protein, followed by transfer onto polyvinylidene difluoride PVDF membranes (Beyotime, Shanghai, China). To assess the expression of target proteins, a chemiluminescence assay was utilized, following previously described methods [19 (link)]. The specific antibodies of CsGGT2 and CsGGT4 used in this study were custom-made in Fanpu Biotechnology Co., Ltd (Fanpu, Wuhan, China). The specificity of these antibodies were detected in ‘Shuchazao’ leaves by western blot (Fig. S4b, and c, see online supplementary material). The internal control in this investigation was the β-actin protein obtained from a plant (Sangon, Shanghai, China).

Ru was purchased from Sigma (St. Louis, MO, USA) and was dissolved in 100% dimethyl sulfoxide (DMSO). A stock solution of 10 mmol/L Ru was prepared and stored as small aliquots at −20 °C for future use. MTT, DMSO, and rat tail collagen type I were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). The primary polyclonal rabbit antibodies (anti-PI3K, anti-AkT, anti-mTOR, and anti-p70S6K) and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were purchased from Elabscience Biotechnology Co., Ltd. (Shanghai, China). Antibodies against β-actin, phospho-specific antibodies (anti-Akt (Ser 473), anti-mTOR (Ser 2448), and anti-p70S6K (Thr 389)) were purchased from Cell Signaling Technology (Danvers, MA, USA). TRIzol reagent, protease inhibitor cocktail, polyvinylidene difluoride (PVDF) membranes, and sodium dodecyl sulfate polyacrylamide electrophoresis (SDS–PAGE) gels were acquired from Beyotime Biotechnology (Haimen, China). Phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). VEGFR2 protein and its ELISA test kits were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cells were washed twice with ice-cold PBS, lysed with 1× radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, China), kept on ice for 30 min, and then centrifuged at 13,000 rpm for 15 min. After protein estimation, sodium dodecyl sulfate (SDS) loading dye was added to the samples, and the samples were boiled at 100°C for 10 min. Clarified lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime Biotechnology, China). The membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (1:1,000) diluted in 2% bovine serum albumin. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:5,000) for 1 h. Signals were detected using a ChemiDoc XRS⁺ chemiluminescence imaging system (Bio-Rad, USA).

Total protein was extracted in RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) supplemented with protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology). Protein concentration was determined using the BCA assay (Thermo Fisher Scientific). Protein samples of 30 μg were separated by SDS-PAGE (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime Biotechnology). The membranes were incubated with anti-mIL-36α (1:1000, R&D systems), anti-mpSTAT3 (1:2000, Cell Signaling Technology, Danvers, MA, United States) and anti-mSTAT3 (1:2000, Cell Signaling Technology) overnight at 4°C, then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at 25 ± 1°C. The protein bands were visualized using BeyoECL Plus (Beyotime Biotechnology).

After treatment with various doses of tryptamine, PC-3 cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 mM PMSF, and the total protein concentration was determined by BCA Protein Assay Kit (Solarbio, Beijing, China). Samples were electrophoresed on a 10% sodium dodecylsulfate (SDS) polyacrylamide gel and electrotransferred onto polyvinylidene difluoride (PVDF) membrane (Beyotime, China). After blocking with 5% BSA for 1 h, the membrane was incubated with the primary antibodies rabbit anti-cleaved caspase-3 (1:1000, ab32042, Abcam) and anti-β-actin (1:1000, 4967S, Cell Signaling Technology, Boston, MA, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000, 7074S, Cell Signaling Technology) for 1 h at 37 °C. After washing with TBST, the bands were visualized by the enhanced chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA) and Odyssey^® imaging system (LI-COR, Lincoln, NE, USA) according to the manufacturers’ instructions.

Total protein of the liver was obtained and measured using an radioimmunoprecipitation (RIPA) buffer including PMSF (1 mmol/L) (Beyotime, Shanghai, China) and a BCA assay kit (Beyotime, Shanghai, China), respectively. Protein extracts were mixed with loading buffer and fully denatured in a boiling water bath according to Yang et al. (2021) with minor changes [24 (link)]. Target proteins were subjected to 8–12% SDS-PAGE electrophoresis, and transferred to a polyvinylidene-difluoride (PVDF) membrane (Beyotime, Shanghai, China). Afterwards, PVDF membranes were washed (3 times × 10 min in 1 × PBST), after blocking 2 h in 5% skim milk. After washing 3 times, PVDF membranes were incubated in primary antibody (GAPDH, NLRP3, and caspase-1) for 8–12 h at 4 °C. Related horseradish peroxidase labeled antibody was incubated at 37 °C for 1 h, after washing 3 times in PBS–0.1% Tween 20 (PBST). Protein bands were quantified and were recorded using the Essential V6 imaging platform (UVITEC, Cambridge, England) with the enhanced chemiluminescence (ECL) chemiluminescence kit (Beyotime Biotechnology). The GAPDH protein served as an internal control protein. The protein expression was expressed as the ratio of band intensities of proteins to that of GAPDH.