Fig 1: SCFFBW7 ubiquitinates surface proteins of cytosolic SPN.(A) Percentage of association of SPN with FBXW7 in A549 cells at 9 hours after infection. n > 100 bacteria per coverslip. (B) Percentage of decoration of SPN with K48-Ub following knockdown of FBXW7, Cullin1, and SKP1 in comparison to scrambled siRNA (siControl) transfected cells. n > 100 bacteria per coverslip. (C) Immunoblot showing ex vivo ubiquitination of BgaA-T in siCullin1-, siFBXW7-, and siSKP1-treated cells. (D) In vitro ubiquitination reaction demonstrating ubiquitination of BgaA-T, BgaA-TK97R, and BgaA-T?Degron. Recombinant active components of SCF complex were incubated with purified BgaA-T-His6 variants, E1, E2 (cdc34), Rbx1, ubiquitin, ATP, and FBXW7. Anti-BgaA and anti-GFP antibodies were used for immunoblotting. (E) Fold change in intracellular survival of SPN in A549 cells transfected with siFBXW7, siCullin1, and siSKP1 and normalized to siControl. (F) Percent association of SPN with K48-Ub in FBXW7R505C-mutated A549 cells. n > 100 bacteria per coverslip. (G) Fold change in intracellular survival efficiency of SPN in FBXW7R505C-expressing A549 cells compared to WT cells. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s test (B and E) and two-tailed unpaired Student’s t test (F and G). **P < 0.01; ***P < 0.005. Data are means ± SD of N = 3 independent biological replicates.