Fig. 5.

CCT knockdown inhibits cell death induced by LF delivered through PA pores in the plasma membrane. (A) Schematic of the assay. (B) Knockdown of CCT in J774A.1 cells was allowed to proceed for 5 d. Graph shows cell survival after LF was delivered through PA pores in the cell membrane. Cells were incubated sequentially with PA and LF at 4 °C, and PA pore formation and translocation were initiated by incubation with pH 5 translocation buffer for 2 min at 37 °C. After translocation, cells were incubated for 4 h at 37 °C in complete medium with 100 nM bafilomycin A1, and cell viability was measured. Incubation at pH 7 does not induce PA pore formation or LF translocation. Data are expressed as mean ± SD of three technical replicates. ****P < 0.0001. (C) Immediately after translocation at pH 5, cells were washed, lysed, and subjected to SDS/PAGE, followed by immunoblot using an anti-PA antibody. cRel was used as a loading control.