Clarity western ecl plus western blotting detection reagent

For protein expression, homogenized tissues were sonicated in ice-cold buffer A (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 μg/ml leupeptin, 2 μg/ml antipain and 1 μg/ml pepstatin; pH 7.4) supplemented with phosphatase inhibitor PhosStop [Roche]. Then, lysates were centrifuged at 1,000×g at 4 °C for 15 min. The supernatant was collected, and protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad, USA) using BSA as standard. Lysate proteins were denatured in SDS-loading dye at 95 °C, separated by SDS-PAGE (10% Tris-glycine), and transferred onto a PVDF membrane (pore size: 0.45 μm; Carl Roth, Karlsruhe, Germany) according to standard protocols. Membranes were blocked with 10% nonfat dry milk (Carl Roth) in 1xTST (50 mM Tris HCl, 0.1% Tween 20, 150 mM NaCl; pH 7.4). Primary antibodies used for protein expression are listed in Supplementary Table 1. Immunoblots were visualized using the Clarity Western ECL plus Western Blotting Detection Reagent (Fisher Scientific) and FUSION FX (VILBER Smart Imaging). Signal intensities were quantified by densitometric analyses using FUSION.Ink software.

Fifteen micrograms of cell lysate protein was denatured in 1x SDS-loading dye at 95°C, separated by SDS-PAGE and transferred onto a PVDF membrane (pore size: 0.45 μm; Carl Roth, Karlsruhe, Germany) according to standard protocols. Membranes were blocked with 10% nonfat dry milk (Carl Roth) in 1xTST buffer (50 mM Tris HCl, 0.1% Tween 20, 150 mM NaCl; pH 7.4) and then incubated with anti-Flag epitope peroxidase-conjugated antibody (1:8,000; A8592, Sigma-Aldrich) or anti-His (1:3,000; 18184, Abcam). GAPDH (1:20,000; 2118S, Cell Signaling, Danvers, MA) was used as a loading control. Immunoblots were visualized using the Clarity Western ECL plus Western Blotting Detection Reagent (Fisher Scientific) and ChemiDoc Touch Imaging System (Bio-Rad).