FIG 2 .

Targeting of GBP1 to S. flexneri is dependent on its functional G domain, its CaaX box, and a C-terminal triple-arginine motif. (A) Schematic depiction of critical protein motifs, domains, and specific residues within the structure of human GBP1. (B) A549 cells were transfected with the mCherry construct (Control), mCherry-tagged wild-type GBP1, and mCherry-tagged GBP1 variants defective in GTP hydrolysis (R48A), nucleotide binding (K51A and S52N), or farnesylation (Δ589–592 and C589A), as indicated. Cells were infected with GFP⁺ S. flexneri at an MOI of 50 and assessed for colocalization with mCherry at 3 hpi inside mCherry-expressing cells. (C and D) HEK 293T cells were transfected with the mCherry construct (Control), mCherry-tagged wild-type constructs (GBP1 and GBP2), and chimeric (GBP1/2^CTSD, GBP1/2^CaaX, GBP2/1^CTSD, GBP2/1^CaaX), deletion mutant (GBP1Δ^PBM), triple mutant (R584–586A), or insertion mutant (GBP2^+PBM, GBP2^{+PBM+GBP1-CaaX}) constructs. Cells were infected with GFP⁺ S. flexneri at an MOI of 50 and assessed for colocalization with mCherry at 3 hpi inside mCherry-expressing cells. CTSD, C-terminal subdomain; PBM, polybasic protein motif. Combined data from 3 independent experiments are shown. Per experiment, >200 bacteria were scored. Error bars indicate SEM. Significance was determined by one-way ANOVA relative to results for GBP1 (B) or as indicated (C and D). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.