Phos stop tablet

Cells were washed with cold PBS, lysed with TX lysis buffer (TXLB; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton-X, 0.5% glycerol, 1% SDS, Complete protease inhibitor [Sigma-Aldrich], and phos-stop tablet [Sigma-Aldrich]), collected by scraping, and transferred into an Eppendorf tube. Samples were boiled for 5 min and sonicated. Protein concentrations were measured using DC Protein assay (Bio-Rad) according to the manufacturer’s protocol, and protein concentrations were normalized by adding TXLB. SDS sample buffer was added on the samples, and samples were boiled for 5 min. To separate the proteins, samples with equal protein content were loaded on precast Tris-Glycine-eXtendet SDS-PAGE gels with a 4–20% gradient (Bio-Rad). The separated proteins were transferred onto nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT. Membranes were incubated in primary antibodies diluted in 5% milk in TBST at 4°C overnight. Membranes were washed three times with TBST and incubated with fluorophore-conjugated Odyssey secondary antibodies (LI-COR Biosciences) for 1 h at RT. Membranes were washed again and scanned with Odyssey infrared system (LI-COR Biosciences). Band intensities were analyzed using ImageJ.