Fig 2. Loss of htrA increases EbpC protein levels in the protoplast fraction.
(A) Immunoblot was performed with α-EbpC on cell wall fractions of WT, ΔhtrA, ΔsrtA, and ΔsrtAΔhtrA strains, as well as ΔsrtAΔhtrA carrying pEmpty (vector control), psrtA, or phtrA. Blot shows typical pilus high molecular weight ladders (HMWL). Relative EbpC density differences were calculated with WT EbpC expression as the standard. (B) Immunoblot was performed with α-EbpC on protoplast fractions of WT, ΔhtrA, ΔsrtA, ΔsrtAΔhtrA, and ΔebpABC strains, as well as ΔsrtAΔhtrA carrying pEmpty (vector control), psrtA, phtrA or phtrAS271A. Top blot shows pilus HMWL and bottom blots show loading and strain controls using α-SecA, α-HtrA and α-SrtA, respectively. Relative EbpC density differences were calculated with WT or ΔsrtA EbpC expression as the standard. (C) Statistical analysis of relative EbpC density from 4 independent immunoblots, using ΔsrtA as the comparison standard, are represented as bar graphs with the standard error of the mean. ** P ≤ 0.01; **** P ≤ 0.0001 (D) Statistical analysis of percent pilus-expressing cells of WT, ΔhtrA, ΔsrtA, ΔsrtAΔhtrA, and ΔsrtAΔhtrA carrying pEmpty (vector control), psrtA or phtrA. Pili were labeled with α-EbpC immune serum and Alexa Fluor 568 secondary antibody. Mean results are represented as bar graphs with standard error of mean. * P < 0.05; ** P ≤ 0.01; *** P≤0.001. Combined data from 5 independent experiments were shown.