V5 tag antibody

Cells were collected by centrifugation (500g for 5 min at RT), washed once with PBS, centrifuged again, and lysed with buffer (140 mM KCl, 10 mM Hepes, 5 mM MgCl₂, 1% Triton X-100, 1 mM TCEP, 2 U/µl Turbo DNase) on ice for 30 min. Lysates were then clarified by centrifugation (20,000g for 10 min at 4°C). Protein lysates were separated on Bolt 4−8% Bis–Tris gels (Thermo Fisher Scientific), then transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk in TBST (0.05% Tween-20) for 1 h at RT. Primary antibodies were incubated overnight at 4°C, and secondary antibodies, for 1 h at RT. C/EBPα protein was probed using a primary C/EBPα antibody (1:1,000, #2295; Cell Signaling Technology), V5 epitope tags were probed by a primary V5-tag antibody (1:2,000, #13202; Cell Signaling Technology), and β-actin loading controls were probed by a primary β-actin conjugated to HRP (1:2,000, #12620; Cell Signaling Technology). An HRP-conjugated anti-rabbit IgG (1:2,000, #7074; Cell Signaling Technology) was used as a secondary antibody against all primary antibodies. All blots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and were visualized by a FluorChem R imaging system (ProteinSimple).