Coomassie brilliant blue r 250

C57BL/6, ICR mice, and SD rats were purchased from Shanghai Jieshige Laboratory Animal Co., Ltd. (Shanghai, China) and housed under specific pathogen-free (SPF) conditions; they had free access to food and water and were under a 12 h light and dark cycle. All experiments involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) and were approved by the Ethics Committee of Shanghai University.
Coomassie brilliant blue R250 (0.025 g, A610037, Sangon Biotech, Shanghai, China) was dissolved in 4.5 mL of methanol and 1 mL of glacial acetic acid and added to 4.5 mL of H₂O. Minoxidil (50 mg, M831481, Macklin, Shanghai, China) was dissolved in 1 mL of glycerol and 1 mL of 75% ethanol and then diluted to the appropriate concentration with 0.9% saline. Ruxolitinib (R849099, Macklin, Shanghai, China) was dissolved in DMSO and then diluted with 20% cyclodextrin. D-arbutin (ID0490, Solarbio, Beijing, China) was dissolved in DMSO and then diluted with 0.9% saline. RSPO1 (50316-M08S) and NBL1 (50976-M08H) proteins were purchased from Sino Biological (Beijing, China). The HaCaT cell line was introduced from the Cell Bank of the Chinese Academy of Sciences (SCSP-5091).

Acrylamide, N,N-methylene bisacrylamide, glycine and tris base were purchased from Jintai Hongda Biotechnology Co., Ltd. (Beijing, China). 2-Thiobarbituric acid, dithiothreitol, thiourea, Coomassie Brilliant Blue R-250, sodium dodecyl sulphate (SDS), bromophenol blue and 2,4-dinitrophenylhydrazine were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). N,N,N,N-tetramethylethylenediamine and β-mercaptoethanol were acquired from Macleans Biochemical Technology Co., Ltd. (Shanghai, China). Coomassie Brilliant Blue kit was provided by Nanjing Jiancheng Institute of Bioengineering (Jiangsu, China). 1,1,3,3-Tetraethoxypropane and chromatography grade formic acid were supplied by Sigma-Aldrich Co., Ltd. (MO, USA). Mass spectrometry-grade trypsin, acetonitrile and water were obtained from Thermo Fisher Scientific (MA, USA). All other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Amylase activity was assayed according to the DNS method. The amounts of released reducing sugars were determined using the dinitrosalicylic acid method [26 (link)]. The absorbance of the reaction system was measured at 540 nm. Up to 1.5 mL of starch solution was added for amylase activity assays, and the reaction was incubated at 40 °C for 10 min. One unit of enzymatic activity (U) was defined as the amount of enzyme needed to release 1 μmol of glucose equivalent per minute. Protein concentration was measured by using a Bradford reagent kit (Sangon Biotech, China). SDS-PAGE was performed using 12% polyacrylamide to determine protein purity. The protein profile was analyzed by staining gels with Coomassie Brilliant Blue R-250 (Sangon Biotech, China) destaining gels with 10% (w/v) acetate solution.

Up to 12.5% gel was prepared for protein separation. The mixture of 15 μL culture supernatant and 3 μL 5× loading buffer was placed in boiling water for 10 min and was loaded into the gel. Coomassie brilliant blue R250 (Sangon, Shanghai, China) was used to color the gel for 30 min. Destainer (glacial acetic acid:ethanol:distilled water, 1:1:8,v/v/v) was used to destain the gel for another 2–4 h.

Shelled raw cashew nut (Anacardium occidentale L.) was purchased from the local HongCheng Market in Nanchang (Nanchang, Jiangxi, China) and stored at −4 °C until use. Gel electrophoresis reagent, molecular weight protein markers (14.4–116.0 kDa) and Folin-phenol reagent were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and Coomassie Brilliant Blue R-250 were supplied by Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals and reagents used were of analytical grade or better.

The expression vector pET28a(+) and E. coli strains BL21 (DE3), were stored at − 80 °C in our lab. Kanamycin, protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and IPTG were purchased from Sangon Biotech (Shanghai, China). p-NPG was purchased from Bio Teke Corporation (Beijing, China). Glycine, Tris, SDS, Bromophenol blue, coomassie brilliant blue R 250 and p-nitrophenol (p-NP) were purchased from San gon Biotech (Shanghai, China). Acrylamide and TEMED were bought from Sigma (USA). Glycerinum, isopropanol, methyl alcohol, β-mercaptoethanol, ammonium persulfate and glacial acetic acid were obtained from Sinopharm Chemical Reagent (Shanghai, China). Thermo Scientific Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (MA, USA). High Affinity Ni-NTA Resin was bought from Genscript (Nanjing, China). All other reagents were of analytical grade and used without any further treatment.

The raw starch-digesting enzyme activity was measured according to the DNS method [42 (link)]. The absorbance of the reaction system was measured at 540 nm. Up to 1.5 mL of starch solution was added for RSDE activity assays, and the reaction was incubated at 40 °C for 10 min. One unit of enzymatic activity (U) was defined as the amount of enzyme required to produce 1 μmol of reducing sugars per min from the reaction substrate. SDS-PAGE was performed using 12% polyacrylamide to determine protein purity. The protein profile was analysed by staining gels with Coomassie Brilliant Blue R-250 (Sangon Biotech, Shanghai, China) and destaining gels with 10% (w/v) acetate solution.