Binding Assay for rJHP0290 Protein

Binding assays were performed as described previously [42 (link)]. Briefly, the cells were treated with Wt or mutant rJHP0290 (2 μg/ml) for 15 min, followed by extensive washing with PBS to remove unbound protein. The cells were incubated with an anti-JHP0290 antibody (1:5,000 dilution) in FACS buffer (2% BSA in PBS) for 1 h on ice, followed by washing with FACS buffer and incubation with an Alexa 488-conjugated anti-rabbit IgG antibody (Molecular Probes) (1:5,000 dilution) in FACS buffer for 30 min on ice. After incubation, the cells were washed twice with FACS buffer and analyzed using an LSRFortessa flow cytometer. FlowJo software (Tree Star, USA) was used for the data analysis.