Journal: Nature immunology

Article Title: RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions

doi: 10.1038/s41590-019-0527-6

Figure Lengend Snippet: (a) Schematic of WT ZAP-S and WT ZAP-L and their CaaX motif (CVIS) mutants generated by site-directed mutagenesis. (b) Confocal immunofluorescence microscopy of Myc-tagged ZAP-S and FLAG-tagged ZAP-L upon co-expression in Huh7 ZAP KO cells. The scale bar represents 5 μm. (c) Super-resolution structured illumination microscopy (SIM) of Myc-tagged ZAP-S and FLAG-tagged ZAP-L co-expressed in Huh7 ZAP KO cells. (d) Confocal immunofluorescence microscopy of tagless WT ZAP-S, WT ZAP-L, or their respective CaaX motif mutants upon expression in Huh7 ZAP KO cells. The scale bar represents 5 μm. (e) Confocal immunofluorescence microscopy of ZAP and LAMP1 upon co-expression of mCherry-LAMP1 and tagless WT ZAP-S and WT ZAP-L, or their CaaX motif mutants. The scale bar represents 5 μm. For (b-e) representative micrographs of three independently performed experiments are depicted. (f) Quantification of co-localization of ZAP with LAMP1-, Rab5-, and Rab7-positive pixels. The correlation coefficients (Pearson’s r) of cells (n=10) from two independent experiments are shown and were calculated using the Fiji Coloc 2 plugin and one-way ANOVA with Tukey’s post-test. Error bars show mean ± SD. *P < 0.01; **P < 0.001.

Article Snippet: Cells were transfected using Trans IT-X2 (Mirus Bio) with expression plasmids encoding respective ZAP isoforms and/or mCherry-tagged organelle markers: Rab5 (Addgene #49201 42 ), Rab7A (Addgene #61804 43 ), and Sec61β (Addgene #49155 43 ) COX8 (Addgene #55102 44 ), PTS1 (Addgene #54520), and LAMP-1 (Addgene #55073). mCherry-tagged Rab5, Rab7A, and Sec61β were gifts from Gia Voeltz and COX8, PTS1, and LAMP-1 were gifts from Michael Davidson.

Techniques: Generated, Mutagenesis, Immunofluorescence, Microscopy, Expressing