Bradford s reagent

The total protein of cell lines was extracted with RIPA lysis buffer (Beyotime) containing protease inhibitor for 20 min on ice. After the measurement of protein concentration by Bradford’s reagent (Beyotime), protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk in TBST for 60 min, membranes were incubated with primary antibodies at 4°C overnight. DRAM1 (sc-81713, Santacruz) and GAPDH (GB11002, Servicebio) were used as primary antibodies. Anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Proteintech) was used as the secondary antibody. Bands were visualized by Bio-Rad Image Lab Software.

The PaCOMT1 cDNA sequence proved to be about 80 bp longer than that of a typical COMT. It harbored two start codons, resulting in two possible transcripts, denoted PaCOMT1-Tr and PaCOMT1. The corresponding open reading frames (ORFs), along with those from PaCOMT2 through 4, were amplified from the four cDNA clones using the gene-specific primer pairs given in Table S3. The resulting amplicons were digested with a number of restriction enzymes (Takara) and ligated into the pET32a vector (Novagen, Darmstadt, Germany) digested with the respective restriction enzymes. The constructs were transformed into E. coli strain BL21 (DE3), and the transgenic cultures were incubated at 37 °C until the OD₆₀₀ reached 0.5. Expression of the transgene was induced by exposure to 1mM (80 μL) isopropyl β-d-1-thiogalactopyranoside (IPTG) for 16 h and a drop in the temperature to 18 °C. N-terminal hexahistidine-tagged proteins were purified by passing through a Ni-NTA Sefinose His-bind column (Bio Basic Inc., Markham, ON, Canada), and then were exchanged through an Ultrafiltration tube (Millipore, MA, USA) in the presence of binding buffer (20 mM Tris–HCl, 500 mM NaCl, pH 8.0). The homogeneity of the heterologously expressed protein was determined by SDS-PAGE and its concentration was assessed using Bradford’s reagent (Beyotime, Shanghai, China) employing bovine serum albumin as the standard.