Flow cytometry analysis of the expression of EP procyclin in control and KIN-E RNAi cells was carried out according to the procedure described previously [37 (link)]. EP procyclin was detected using the monoclonal antibodies TRBP1/247 (Cedarlane Labs, Canada) [38 (link)], which was used at a dilution of 1:500, and the FITC-conjugated anti-mouse IgG (Sigma-Aldrich), which was used at a dilution of 1:400. Cells were analyzed on a fluorescence-activated cell sorter (Becton Dickinson & Co., Sunnyvale, CA).
For quantitative western blotting, an equal number (5×105) of control and KIN-E RNAi cells were lysed, and cell lysate was fractionated on SDS-PAGE, transferred onto a PVDF membrane, and immunoblotted with anti-EP procyclin monoclonal antibody TRBP1/247 (1:1,000 dilution, Cedarlane Labs) and anti-TbPSA6 polyclonal antibody (1: 2,000 dilution), which detects the alpha-6 subunit of the 26S proteasome [39 (link)], for 1 hr at room temperature. After washing three times with TBST, the membrane was incubated with FITC-conjugated anti-mouse IgG (1: 400 dilution, Sigma-Aldrich) and IRDye 680LT anti-rabbit IgG (1:2,500 dilution, Li-Cor Cooperate), and western blot signals were captured using the Bio-Rad ChemiDoc MP imaging system, which allows multiplex fluorescent western blot imaging and quantitative analysis of protein bands.
For quantitative western blotting, an equal number (5×105) of control and KIN-E RNAi cells were lysed, and cell lysate was fractionated on SDS-PAGE, transferred onto a PVDF membrane, and immunoblotted with anti-EP procyclin monoclonal antibody TRBP1/247 (1:1,000 dilution, Cedarlane Labs) and anti-TbPSA6 polyclonal antibody (1: 2,000 dilution), which detects the alpha-6 subunit of the 26S proteasome [39 (link)], for 1 hr at room temperature. After washing three times with TBST, the membrane was incubated with FITC-conjugated anti-mouse IgG (1: 400 dilution, Sigma-Aldrich) and IRDye 680LT anti-rabbit IgG (1:2,500 dilution, Li-Cor Cooperate), and western blot signals were captured using the Bio-Rad ChemiDoc MP imaging system, which allows multiplex fluorescent western blot imaging and quantitative analysis of protein bands.