Ha tag rabbit polyclonal antibody

The cellular thermal shift assay (CETSA), as described previously (Guo et al., 2020 (link)), was employed to evaluate the binding of genistein and viral RPO30 protein in living cells. In brief, HEK293 cells were, respectively, transfected with pCMV-N-HA-ORFV RPO30 or pCMV-N-HA-ORFV RPO35 plasmid using LipoFiter 3.0 (Hanbio, China) for 40 h and exposed to 100 μM genistein or DMSO at 37°C with 5% CO₂ for 2 h. Subsequently, the cells were washed with PBS to remove excess drugs, harvested, and then lysed on ice for 30 min in RIPA lysis buffer. Protein concentrations were determined by the BCA protein quantification assay (Beyotime, China). Protein solutions were adjusted to 1 mg/mL, and 30 μL aliquots of supernatants were added to a Thermal Cycler (Bio-Rad, USA), heated at different temperatures for 5 min, and then chilled at room temperature for 3 min. After centrifugation at 13,000×g for 30 min at 4°C, supernatants were transferred to new tubes, separated by SDS-PAGE on 10% gels, transferred to PVDF membrane, and incubated with primary antibodies HA tag rabbit polyclonal antibody (Proteintech, China) or GAPDH/β-actin mouse monoclonal antibody (Proteintech, China) and appropriate secondary antibodies.

For K63-linked polyubiquitination assays, HA-ubiquitin-K63 (all other Lys residues were mutated to Arg except K63) and myc-MavC or its mutants were cotransfected into cells. For all western blotting assays, HEK293T cells were collected at 28 h posttransfection and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% NP40, and 1% SDS). Cell lysates were resolved by SDS-PAGE gels and transferred to PVDF membranes for immunoblotting analysis with specific antibodies: mouse monoclonal Flag-specific antibody M2 (Sigma-Aldrich, Cat# F3165), 1:10,000; mouse monoclonal myc-specific antibody 9E10 (Santa Cruz Biotechnology, Cat# SC-40), 1:5000; mouse monoclonal β-actin-specific antibody (Sigma-Aldrich, Cat# a5316), 1:10,000; HA tag rabbit polyclonal antibody (Proteintech, Cat# 51064-2-AP), 1:5000; UBE2N (D2A1) rabbit mAb (Cell Signaling Technology, Cat# 6999S), 1:3000.