Topbio multigel 21 chemiluminescent imaging system

After the indicated treatment, cells were collected and lysed to determine the protein expression. Cells were lysed in T-PER tissue protein extraction buffer to obtain total cell extract or NE-PER™ nuclear and cytoplasmic extraction reagents to isolate nuclear fraction. Collected protein was used for SDS-polyacrylamide gel electrophoresis, and protein concentration was determined using the Bio-Rad protein assay kit (Hercules, CA, USA). The membranes were incubated, each with indicated primary antibodies by different dilutions overnight at 4 °C. Membranes were then washed with TBST and incubated with secondary antibodies (diluted 1:10,000 in 5% nonfat milk), shaking at room temperature for 1 h. Protein expression was detected with chemiluminescence using an Amersham ECL detection kit (Cytiva, Marlborough, MA, USA), and the signal was captured using the TopBio Multigel-21 chemiluminescent imaging system (Topbio, Taiwan) and quantified with the ImageJ software version 1.54 (National Institutes of Health, Bethesda, MD, USA).

Cells were lysed in T-PER tissue protein extraction buffer to obtain total cell extract or NE-PER nuclear and cytoplasmic extraction kit to isolate nuclear fraction. Protein concentration was measured by Bradford protein assay. Then, proteins were fractionated by SDS-PAGE, and the separated proteins were transferred onto PVDF membranes. Membranes were blocked in 5% nonfat milk in TBST buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) with 1 h shaking at room temperature. The blot was probed with primary antibodies (diluted 1:1000 in 2% BSA) overnight at 4 °C. Subsequently, membranes were washed with TBST buffer and hybridized with secondary HRP-conjugated antibody (diluted 1:10000 in 5% nonfat milk) with shaking at room temperature for 1 h. Protein expression was detected with chemiluminescence using an Amersham ECL detection kit, and the signal was captured using the TopBio Multigel-21 chemiluminescent imaging system (Topbio, Taiwan) and quantified with the ImageJ software (National Institutes of Health, Bethesda, MD).