Anti gapdh antibody sc 32233

MIN6 cells were transfected with siRNA as described above, and transfected MIN6 cells were then washed with phosphate-buffered saline (PBS); isolated mouse islets or homogenized lung were suspended in lysis buffer [10 mM Tris⋅HCl (pH 7.4)/100 mM NaCl/1% (wt/vol) SDS] containing protease inhibitor cocktail (Roche, Germany). Thirty micrograms of total protein were subjected to SDS-PAGE gels (Bio-Rad, USA), transferred to nitrocellulose filters, and subjected to immunoblot analysis. After blocking with PBS containing 0.1% Tween 20 and 5% skim milk at room temperature for 1 h, blotted membranes were incubated overnight at 4 °C with anti-IP3R1 antibody (8568; Cell signaling, USA) at 1:1000 dilution, anti-IP3R3 antibody (610312; BD Biosciences, USA) at 1:1000 dilution, anti-GAPDH antibody (SC-32233; Santa Cruz, USA) at 1:1000 dilution. Blotted membranes were then washed by PBS containing 0.1% Tween 20 and incubated with anti-rabbit or anti-mouse IgG secondary antibody (GE Healthcare, UK) diluted at 1:3000 at room temperature for 30 minutes before detection using ECL prime (GE Healthcare, UK). Band intensity was quantified with Multi Gauge software (Fujifilm, Japan).