Sds running buffer

Cells of the human oral cancer cell lines, DOK, and HOK were rinsed with phosphate buffered-saline (PBS; Sigma–Aldrich, St Louis, MO, USA) and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma–Aldrich). The lysates were subsequently centrifuged at 4 °C, 14,000 rpm, for 15 min. Equal amounts of protein were denatured by adding sodium dodecyl sulfate (SDS) running buffer (Sigma–Aldrich) and β-mercaptoethanol (Sigma–Aldrich). The samples were then analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma–Aldrich) on 15% gels, and the proteins were transferred onto a poly vinylidene fluoride (PVDF) membrane (Sigma–Aldrich) using Bio-Rad's transblot with the primary rabbit polyclonal primary anti-Orai1 (Invitrogen; 1: 1000) and anti-STIM1 (Invitrogen; 1: 1000) antibody respectively with species specificity for human tissues and observed molecular weight 55 kDa for Orai1 and 75 kDa for STIM1 respectively, and β-actin (Sigma–Aldrich; 1: 500), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma–Aldrich; 1: 5000). The amount of protein was then quantified using a Fuji LAS-4000 lumino image analyzer (Fuji Photo Film Co., Tokyo, Japan). The ratio was normalized by the β-actin signal.

Cells of human oral cancer cell lines (Ca9-22, and OECM-1), DOK, and HOK were rinsed with phosphate buffered-saline (PBS; Sigma–Aldrich, St Louis, MO, USA) and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma–Aldrich). The lysates were subsequently centrifuged at 4 °C, 14,000 rpm, for 15 min. The protein concentrations were measured using a Thermo Pierce Protein Assay Kit. Equal amounts of protein were denatured by adding SDS running buffer (Sigma–Aldrich) and β-mercaptoethanol (Sigma–Aldrich). The samples were then analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma–Aldrich) on 15% gels, and the proteins were transferred onto a poly vinylidene fluoride (PVDF) membrane (Sigma–Aldrich) using Bio-Rad's transblot with the primary rabbit polyclonal anti-TRPM6 antibody (Abnova; Cat. no. PAB3252, 1: 500), with species specificity for human tissues and observed molecular weight 171 kDa, and β-actin (Sigma–Aldrich; 1: 5000), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma–Aldrich; 1: 5,000). The amount of protein was then quantified using a Fuji LAS-4000 lumino image analyzer (Fuji Photo Film Co., Tokyo, Japan). The ratio was normalized by the β-actin signal.