The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T–mediated virulence
J. Clin. Invest. Priya Balachandran, et al. 117:419 doi:10.1172/JCI28792 [Go to this article.]

Figure 3
Role of Crk and Cbl-b in the ubiquitination and degradation of ExoT. (A–E) HeLa cells were pretreated with (A) no siRNA, (B and E) control siRNA, (C) Cbl-b siRNA, or (F) Crk siRNA for 30 hours prior to infection with PA103ΔexoU/exoT(G-A+). Translocation assays were performed as described for Figure 1. Cytoplasmic extracts were analyzed by immunoblotting using Abs against ExoT (A–C, E, and F; top panels) and loading control GAPDH (A–C, E, and F; middle panels). siRNA knockdown efficiency was determined by immunoblotting against Cbl-b (A–C; bottom panels) or Crk (E and F; bottom panels). (D) Rates of degradation in the presence or absence of Cbl-b siRNA were quantified as described for Figure 1C and plotted as means ± SD. Error bars are too small to be seen at some points. (G–H) HeLa cells were pretreated with (G) vector control pCEFL or (H) pCEFL–HA–Cbl-b (C373A) for 24 hours. Translocation assays were performed as described for Figure 1. Cytoplasmic extracts were analyzed by immunoblotting using Abs against ExoT (top panels) and GAPDH (middle panels). Blots were probed with anti-HA Abs (bottom panels) to verify expression of transfected protein. (I) HeLa cells were transfected with pCruzMycB-ExoT(G-A+), pcDNA3-(HA-Ub)⁴ and pCEFL, pCEFL–HA–Cbl-b, or pCEFL–HA–Cbl-b (C373A) for 18 hours. Ubiquitination of ExoT was assessed as described for Figure 1D. ExoT was immunoprecipitated using anti-ExoT antiserum under conditions in which ExoT and Cbl-b do not coimmunoprecipitate (in the presence of 0.5% SDS) and immunoblotted with anti-HA (top panel, ubiquitinated ExoT) or anti-Myc (bottom panel, total ExoT).