Fig. 4: Rof regulates the virulence program of Salmonella Typhimurium.

Western blotting analysis of total protein levels or secreted proteins in WT Salmonella and Δrof mutants. The secreted proteins are extracted from the culture supernatant. pEV, empty vector. pRof, Rof complementation plasmid, contains a copy of rof gene with its own promoter. Total proteins are separated by 12% SDS-PAEG and analyzed using anti-FLAG antibody. GroEL serves as a loading control. Representative data from two independent experiments are shown. Uncropped images are provided as Source Data file. a–c Western blotting analysis of HilA-3xFLAG protein levels (a); PrgH-3xFLAG protein levels (b); SopB-3xFLAG protein levels (c) in total cellular proteins. d–e Western blotting analysis of SopB-3xFLAG protein levels (d) and SipC protein levels (e) in total proteins and secreted proteins. GroEL served as a non-secreted cellular protein control. f Western blotting analysis of PrgH-3xFLAG levels in total bacterial proteins. pEV, empty vector. pRof, Rof complementation plasmid, contains a copy of rof gene with its own promoter. The indicated point mutations were introduced in pRof. g Rof is required for Salmonella invasion of epithelial cells. Human HCT-116 cell line was incubated with WT Salmonella and rof mutants using an MOI of 10. Gentamicin was added to kill extracellular bacteria after 40 min post-infection. The intracellular bacteria were harvested and enumerated after serial dilution and plating on LB agar. Data are presented as mean values ± SD from n = 3 biological replicates.