Figure 4.

PAD2 and PAD4 directly bind P. aeruginosa. Flow cytometry analysis of PAD2 (A, B) and PAD4 (C, D) (10–80 nM) membrane interaction with P. aeruginosa (5 × 10⁷ CFU) after 30 min of incubation at 37°C. rPAD2 and rPAD4 binding was evaluated using anti-PAD2 and anti-PAD4 rabbit polyclonal antibodies, respectfully, followed by use of anti-rabbit PE-conjugated antibody. Data were analyzed by one-way ANOVA followed by multiple comparison Tukey’s post-hoc test and presented as mean ± SEM, n = 3 biological replicates. (E) Subcellular fractions of P. aeruginosa [bacterial whole-cell lysate (L), cytosol (C), and membrane proteins (M)] were electrophoresed on a polyacrylamide gel and stained with Coomassie Blue (top panel). A Western blot for P. aeruginosa LPS confirmed purity of membrane proteins (lower panel). (F) The subcellular distribution of the bacterial adhesion to which PADs bound was determined after transfer to PVDF membrane and incubation with rPAD2 or rPAD4. Arrows indicate protein bands of ∼60, 42, and 26 kilodaltons in the outer membrane fractions corresponding to molecules with which rPAD2 and rPAD4 interacted.