Fig. 3: Internalized Als3 acts through caspase-8 to regulate immune responses.

a Western blot analysis of pull-down products from LPS-primed BMDMs transfected with Als3-His or Als3^S70Y-His for 1.5 h using Profect-P2 transfection reagent. WCL, whole cell lysates. b BMDMs were incubated with Alexa Fluor 488-conjugated Als3 or Als3^S170Y at 37 °C for 1 h, fixed, and then observed under confocal microscopy. An antibody against full-length caspase-8 was used to stain the samples. Arrows at images indicate examples of colocalization of Alexa Fluor 488-conjugated Als3 with caspase-8. Scale bars, 5 μm. c Immunofluorescence images of BMDMs after infection with hyphae for 2 h in the presence of 50 mM KCl. Scale bars, 5 μm. Antibodies against Als, procaspase-8, and ASC were used to stain the samples. d Western blot analysis of the indicated markers for inflammasome activation after infection with hyphae. Results are representative of two individual experiments. e Western blot analysis was conducted on both WT and Casp8^-/- 293 T cells. These cells were subjected to overexpression of FLAG-tagged mouse ASC (FLAG-ASC) for 14 h, followed by infection with the hyphal form of either the als3^∆/∆::ALS3 strain or the als1^SY3^SY5^∆/∆ mutant for 3 h. Results in (a, d, e) are representative of three independent experiments. Images in (b, c) are representative of three individual experiments with at least six randomly selected fields for each experiment.