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. Author manuscript; available in PMC: 2008 Nov 1.
Published in final edited form as: Traffic. 2008 Aug 4;9(11):1948–1957. doi: 10.1111/j.1600-0854.2008.00808.x

Figure 1. P. aeruginosa ExoS inhibition of bacterial internalization by HeLa cells is RhoGAP dependent.

Figure 1

HeLa cells were intoxicated with P. aeruginosa expressing ExoS derived constructs. A) Three hr post intoxication (pi), cells were collected, lysed in SDS-PAGE sample buffer, and subjected to SDS-PAGE and immunoblotted for HA, using Super Signal detection. GAPDH was also probed as a loading control. B) Three hr pi, cells were incubated with 300 μg/ml of Gentamycin for 1 hr to kill extracellular bacteria. Cells were then collected, washed and lysed in 1% Triton X-100 to release intracellular bacteria. Lysates were diluted serially, plated on LB agar to quantify the CFU of internalized bacteria. HeLa cells were pretreated with nocodazole (noco) or cytochalasin D (cytoD) for 0.5 hr, then infected with catalytic null pUCP-ExoS(R146K, E381/379D)-HA (G−A−) as negative or positive controls, respectively. Results were normalized with total cell number by LDH activity and reported as the average of three independent experiments. Constructs used: pUCP vector control (pUCP), catalytic null pUCP-ExoS(R146K, E381/379D)-HA (G−A−), wild type pUCP-ExoS-HA (G+A+), GAP only pUCP-ExoS-E381D-HA (G+), ADPr only pUCP-ExoS-R146K-HA (A+), truncated ExoS with GAP only pUCP-ExoS-1-234-HA, *, p<0.05, **, p<0.01.