Trans blot sd semi dry transfer blotter

Western blotting was performed as described previously^{15 (link)}. The proteins separated with SDS-PAGE were transferred to PVDF membranes (FluoroTrans, Pall Corp., Port Washington, NY) using a Trans-Blot SD Semi-Dry Transfer blotter (Bio-Rad Laboratories, Hercules, CA), then incubating the membranes with 5% (w/v) skim milk in TBS for 1 hour and washing three times with T-TBS (TBS containing 0.02% Tween 20). The membranes were then incubated with primary antibodies overnight at 4 °C, washed three times with T-TBS, then incubated with horseradish peroxidase–conjugated IgGs for 1 hour at room temperature. The membranes were washed three times with T-TBS and stained with Clarity Western ECL Substrate (Bio-Rad) according to the manufacturer’s instructions and visualized using a ChemiDoc Touch (Bio-Rad).

Western blotting was performed by separating the proteins with SDS-PAGE, transferring the proteins to nitrocellulose membranes (Amersham Protran, GE Healthcare, Chicago, IL) using a Trans-Blot SD Semi-Dry Transfer blotter (Bio-Rad Laboratories, Hercules, CA), then incubating the membranes with 5% (w/v) skim milk in TBS for 1 h and washing three times with T-TBS (TBS containing 0.02% Tween 20). Membranes were then incubated with primary antibodies overnight at 4 °C, washed three times with T-TBS, then incubated with horseradish peroxidase–conjugated IgGs for 1 h at room temperature. Membranes were washed three times with T-TBS and stained with Clarity Western ECL Substrate (Bio-Rad) according to the manufacturer’s instructions and visualized using a ChemiDoc MP (Bio-Rad).

The expression vectors of HA-tagged CPT1 or HA-tagged CEPT1 described previously were transfected into DKO cells using Lipofectamine 2000. After 24 h, the cells were lysed in 20 mM Tris-HCl buffer, pH 8.0, containing 0.2% Triton X-100 (w/v) and proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Proteins were separated with SDS-PAGE, transferred to PVDF membranes (FluoroTrans; Pall Corp., Port Washington, NY) using a Trans-Blot SD Semi-Dry Transfer blotter (Bio-Rad Laboratories, Hercules, CA), and then the membranes were incubated with 5% (w/v) skim milk in TBS for 1 h. The membranes were then incubated with anti-HA tag (Cell Signaling Technology) and anti-GAPDH (Wako Pure Chemical Industries) antibodies overnight at 4°C, washed three times with TBS containing 0.1% Tween-20 (w/v), and then incubated with horseradish peroxidase-conjugated IgGs for 1 h at room temperature. The membranes were washed three times with TBS containing 0.1% Tween-20 and stained with Clarity Western ECL Substrate (Bio-Rad) according to the manufacturer's instructions and visualized using a ChemiDoc Touch imaging system (Bio-Rad). Protein density was quantified using Image Lab software (Bio-Rad). Results were normalized against the density of GAPDH.