Hybond p polyvinylidene fluoride membrane

Western blot analyses were performed utilizing whole-cell lysates. Briefly, cells were lysed by incubating for 30 min on ice in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), containing proteinase and phosphatase inhibitors. Cell debris was removed via centrifugation at 10,000 g for 10 min at 4 °C. The protein concentration of cell lysates was determined using the Bradford method (Bio-Rad). Proteins (100 μg) in cell lysates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 7% gel with or without 2-ME and then transferred to a Hybond-P polyvinylidene fluoride membrane (Amersham Biosciences). The membrane was first incubated with primary antibodies against IP₃R1 (Cell Signaling Technology), phospho-IP₃R1 (Cell Signaling Technology), IP₃R2 (Merck Millipore), IP₃R3 (BD Biosciences) and α-Tublin (Santa Cruz Biotech), and then with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence reagents (Amersham Biosciences).