Anti his tag

The library of natural product collection was obtained from MicroSource Discovery systems Inc. The 96-well plates for bioluminescent signal detection were purchased from PerkinElmer Life Sciences. Coelentarazine 400a was from Gold Biotechnology. Anti-His tag, anti-β-actin and anti-green fluorescent protein (GFP) antibodies were from Gene Tex. Anti-CPR and anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were from Abcam. Anti-GFP-affinity Sepharose was obtained from Abcam. Danthron (1,8 dihydroxyanthraquinone), 1,4,5-trihydroxyanthra-9,10-quinone, 1-hydroxyanthra-9,10-quinone, and anthrarufin (1,5-dihydroxyanthraquinone) were purchased from Sigma-Aldrich.

HEK293T cells transfected with pTriEx-mPB and pTriEx-NP-mPB were washed twice with ice-cold PBS and lysed in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 10 µg/ml aprotinin, and 50 µM ρ-APMSF). The lysates were centrifuged, and the protein extracts were separated on 8% SDS-PAGE and transferred to Immobilon transfer membranes (PVDF) (Millipore, Bedford, MA, USA). The membranes were washed, incubated in TBST containing 5% skim milk for 1 h, and treated overnight with an anti-His tag (1∶2000; GeneTex, Inc., Irvine, CA, USA), anti-GFP (1∶5000; GeneTex, Inc.), or anti-α-tubulin antibody (1∶5000; Abcam plc, Cambridge, UK) at 4°C. A horseradish peroxidase-conjugated secondary antibody (1∶5000; GeneTex, Inc.) was applied to allow color reaction for detection. For nucleo-cytoplasmic separation experiments, cytoplasmic and nuclear protein fractions were extracted from transfected HEK293T cells with the ProteoJET cytoplasmic and nuclear protein extraction kit (Thermo Fisher Scientific Inc.).