Fig 6. Arf6 is recruited by R. typhi RalF and is required for infection.
(A) Ectopically expressed RalFRtFL co-localizes with Arf6 but not Arf5. HeLa cells co-expressing EYFP, EYFP-RalFRtFL or EYFP-RalFRbFL and mRFP-Arf6 (left) or -Arf5 (right) were fixed with 4% para-formaldehyde. Nuclei were stained with DAPI (blue). (Scale bar: 10 μm). (B) RalFRtFL pull-down of Arf6. Lysates from HEK293T cells expressing mRFP-Arf5 or -Arf6 were incubated with HisPur Cobalt resin bound with rHis-RalFRtFL or resin alone. Bound proteins were eluted with imidazole and analyzed by protein immunoblot using antibodies as indicated. (C) Arf6 is recruited during R. typhi entry. HeLa cells expressing mRFP-Arf5 or -Arf6 (red) were infected with partially purified R. typhi (MOI ~100). Ten minutes post infection, cells were fixed and R. typhi detected with anti-R. typhi serum (green). DAPI (blue) is shown in the merged image. Boxed regions are enlarged to show detail. White arrowheads indicate R. typhi. (Scale bar: 5 μm). (D) Arf6 knockdown inhibits R. typhi infection. HeLa cells transfected with negative, Arf6, or Arf5 siRNA were infected with partially purified R. typhi (MOI ~100). At 2 hrs post infection, cells were fixed, plasma membrane stained with Alexa Fluor 594 wheat germ agglutinin, and R. typhi detected with rat anti-R. typhi serum and Alexa Fluor 488 anti-rat antibody. The number of R. typhi per host cell was counted for 100 host cells for three independent experiments. Error bars represent mean ± SD (Student’s two-sided t-test). (Scale bar: 5μm). (E) Confirmation of Arf6 and Arf5 knockdown. Arf6 and Arf5 knockdown, 80% and 96% respectively, was confirmed by western blot and densitometry analysis using ImageJ (NIH).