Beyogel plus precast page gel

The whole cell lysate samples were run on BeyoGel™ Plus Precast PAGE Gel (4 to 10%) for Tris-Gly System (P0465S, Beyotime) for 110 min at 110 V. Gels were transferred to PVDF membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked by 5% (w/v) non-fat milk (A600669-0250, Sangon Biotech) at 4 °C overnight, before sliced into stripes according to a pre-stained ColorMixed Protein Marker (PR1920, Solarbio). The sliced stripes were incubated with primary antibody for 1 h at ambient temperature. β-actin were used to be a reference protein for Western blot in this project. Then the membranes were washed five times with TBST buffer before incubation with secondary antibody for 30 min at ambient temperature. The membranes were washed three times and imaged via ImageQuant LAS4000 (Cytiva). For semi-quantitative analysis, band intensities were measured by densitometry using the software ImageJ 1.38X. Primary antibodies used in this study include: FLAG tag mouse monoclonal antibody (1:20,000) (66008-3-Ig, Proteintech) and β-actin mouse monoclonal antibody (1:5000) (66009-1-Ig, Proteintech). Secondary antibody used in this study is peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:10,000) (SA00001-1, Proteintech).

The whole cell lysate samples expressing ABCG25_WT or mutants were run on BeyoGel Plus Precast PAGE gel (8–20%) for the Tris-Gly system (Beyotime) for 120 min at 100 V. The gel was transferred to a polyvinylidene fluoride membrane (Merck Millipore) with the Trans-Blot SD semi-dry electrophoretic transfer system (BioRad). The membrane was blocked with 5% (w/v) non-fat milk (Sangon Biotech) for 1 h at room temperature, followed by incubation with the primary antibody (anti-FLAG tag mouse monoclonal antibody, 1:3,000, CWBIO) for 1 h at room temperature. Then the membrane was washed 5 times with TBST buffer (20 mM Tris-HCl, pH 7.0, 150 mM NaCl, 0.1% (w/v) Tween-20) before incubation with the secondary antibody (HRP-conjugated goat-anti-mouse IgG, 1:5,000, CWBIO) for 1 h at room temperature. The membrane was washed 5 times and imaged after incubation with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific).