Fig 2: ASC is recruited to the site of dTGN-NLRP3. HeLa cells expressing (a) NLRP3-GFP or (b) GFP were infected with Sk02 at an MOI of 10. At indicated time points post-infection, cells were subjected to immunofluorescence for NLRP3 (green), TGN46 (red), and IAV (cyan). Scale bar, 25 μm. HeLa cells expressing NLRP3-GFP (c) or GFP (d) were transfected with a plasmid expressing ASC-Myc for 24 h and then were infected with Sk02 at an MOI of 10. At indicated time points, the cells were subjected to immunofluorescence microscopy for NLRP3 (green), TGN46 (red), ASC (blue), and IAV (cyan). The white arrows indicate the co-localization of NLRP3 and ASC. Scale bar, 50 μm.

Fig 3: Immunofluorescence confocal images showing Rab10 and pRab10 expression in subcellular compartments in the C57BL/6J WT mouse cortex. a Rab10 (green) colocalized with the endoplasmic reticulum (ER) marker, KDEL (red) shown in merged and zoomed in images. Scale bar = 10, 2 µm. b pRab10 (green) did not show colocalization with the ER marker, KDEL (red) shown in merged and zoomed in image. Scale bar = 10, 2 µm. c Colocalization analyses using Mander’s colocalization coefficient (MCC) indicated a significant difference Rab10 protein colocalization with KDEL compared to pRab10 (p value = 0.001). d Rab10 (green) colocalized with the trans Golgi network (TGN) marker, TGN46 (red) shown in merged and zoomed in image. Scale bar = 10, 2 µm. e pRab10 (green) did not show colocalization with TGN46 (red) shown in merged and zoomed in image. Scale bar = 10, 2 µm. f Colocalization analyses using MCC indicated a significant difference in Rab10 protein colocalization with TGN46 compared to pRab10/TGN46 (p value = 0.01). g Rab10 (green) colocalized with the lysosomal marker, LAMP1 (red) shown in merged and zoomed in image. Scale bar = 10, 2 µm. h pRab10 (green) did not show colocalization with LAMP1 shown in merged and zoomed in image. Scale bar = 10, 2 µm. i Colocalization analyses using MCC indicated a significant difference in Rab10 protein colocalization with LAMP1 compared to pRab10 (p value = 0.004) j Rab10 (green) did not show colocalization with the early endosome marker, EEA1 (red) shown in merged and zoomed in image. Scale bar = 10, 2 µm. k pRab10 (green) did not show colocalization with EEA1 shown in merged and zoomed in image Scale bar = 10, 2 µm. l There was no significant difference between Rab10 and pRab10 colocalization signal with EEA1 indicated by MCC (p value = 0.4). Colocalization analysis was performed using ImageJ plug in, JACoP, and unpaired t-test with Welch’s correction was used to get the significance value. (n = 3 biologically independent mouse brain samples)