Immunofluorescence Analysis of CARDS Toxin

Immunofluorescence analysis (IFA) was performed as described previously^{20 (link)}. Briefly, HeLa cells (2 × 10⁴ cells/well) grown on glass coverslips treated with or without specific pharmacological agents and CARDS toxin (140 pmol) or C-CARDS (70 pmol) were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton-X-100 and blocked with 1% normal goat serum (NGS; Gibco). Then, cells were washed with 0.2% NGS and treated with rabbit polyclonal anti-CARDS toxin (1:1000) as indicated previously and incubated with secondary goat polyclonal anti-rabbit antibody (1:1000 dilution) labeled with Alexa Fluor 555 (Invitrogen) for 1 h. Cellular F-Actin was stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen). For co-IFA studies, cells were incubated with GM130 (1:1000, Abcam) or ERGIC (1:100; Santa Cruz) monoclonal antibodies in PBS with 0.2% NGS in PBS for 1 h. Cells were washed with PBS containing 0.2% NGS and incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse, 1:500) in PBS with 0.2% NGS for 1 h at RT. Individual samples were mounted in medium containing 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories;), and images were acquired using a Carl-Zeiss immunofluorescence microscope, and Z sections were prepared using AxioVision deconvolution software and enhanced in Adobe Photoshop.