Primary antibody dilution buffer

The concentration of recombinant proteins (rPoMSP4) was determined through the Bradford method using bovine serum albumin (BSA) as standard (Bradford protein assay kit, Solarbio). Purified proteins were analysed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Beyotime Biotech) to assess the expression level and immunoreactivity. The separated proteins from SDS-PAGE were electrophorectically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon) and blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk at 4 °C. The membranes were probed with anti-His antibody (ABclonal) at 1:5000 dilution along with primary antibody dilution buffer (Meilunbio) overnight at 4 °C. Membranes were washed three times with 0.1% TBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for 90 min. Finally, the membranes were analysed with a ChemiDoc MP imaging system (Bio-Rad).

Cells were lysed in RIPA Lysis Buffer (MA0151, Dalian Meilun Biotechnology Co., Ltd., China) containing a protease inhibitor cocktails (FD1001, Fudebio, Hangzhou, China) on ice. Equal amounts of total protein from different samples were separated by SDS-PAGE gels at 100 V for 1.5 h and transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ) at 280 mA for 1.5 h. Then, the membranes were blocked with 5% skim milk powder in TBST for 1 h at room temperature and treated with specific primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China). Each band was detected using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Anti-GAPDH, anti-CREB3, anti-Bcl-2, anti-mmp2, anti-Vimentin and anti-E-cadherin antibodies were purchased from Abcam (Cambridge, UK). The anti-c-Jun, anti-cleaved-caspase 3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); anti-mmp9 antibody was purchased from ABclonal (Boston, MA, USA); and anti-N-cadherin antibody was purchased from Proteintech (Chicago, USA). Primary Antibody Dilution Buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (MB9881, Dalian, China).