P nitrophenyl α d galactopyranoside

Mannobiose (M₂), mannotriose (M₃) and mannotetraose (M₄) standards were purchased from Megazyme (Bray, Ireland). Locust bean gum (LBG), solka floc, glucose, mannose, guar gum, p-nitrophenyl-α-d-galactopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-mannopyranoside, p-nitrophenol (pNP) and other chemicals were sourced from Sigma-Aldrich, USA. Copra meal was obtained from Parker Biotech Private Ltd., Tamil Nadu, Chennai, India. Food-grade konjac gum (glucomannan) was obtained from New Foods, Bloomingdale, Illinois, USA. Fenugreek seed (Trigonella foenum-graecum) meal, Aloevera pulp, rice husk, wheat straw and wheat bran were purchased from local markets.

Extracellular α‐galactosidase assay was performed to determine the expression level of the Mel1p, as described previously (Diep et al. 2006 (link)). Yeast strains were grown in synthetic complete medium containing gly/lac up to saturation. The cultures were then subcultured in synthetic complete media containing 2% galactose to an initial OD of 0.05. The cultures were then allowed to grow till an OD of 1.00. A volume of 1 mL of each culture was centrifuged and extracellular α‐galactosidase activity of the supernatant was determined as follows. A total of 120 μL of the supernatant was mixed with 360 μL of assay buffer (2 volumes of 0.5 m sodium acetate, pH 4.5, and 1 volume of 100 mm p‐nitrophenyl α‐d‐galactopyranoside [Sigma]). The reaction was incubated at 30°C for 5 h and terminated by adding 520 μL of stop buffer (1 M sodium carbonate). Enzyme amounts were then determined by measuring the absorbance at 410 nm. Triplicate samples were taken for the analysis and results represent average of at least three independent experiments with standard deviation.

The restriction enzymes were obtained from New England Biolabs (Beijing, China). p-nitrophenyl α-D-galactopyranoside, guggulsterone, CDCA, yeast nitrogen base without amino acids, dimethyl sulfoxide (DMSO) and glucose were all purchased from Sigma (Shanghai, China). The dropout supplement free from leucine and tryptophan (-Leu/-Trp DO supplement) and quantitative real-time PCR kit were bought from Takara (Dalian, China). Dulbecco's modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) was from Gbico (Shanghai, China). 293Fect was purchased from Pregene (Beijing, China). Dual-Luciferase Reporter Assay System was obtained from Promega (Beijing, China). RNA extraction reagent and reverse transcription kit were purchased from Toyobo (Shanghai, China). Rabbit anti-phosphorylated-STAT3, rabbit anti-total-STAT3 and caspase9 were from Bioworld Technology, Inc (Nanjing, China). GAPDH antibody was obtained from Kangcheng Bio-tech (Shanghai, China). All NSAIDs were purchased from Sigma.