Fig. 2

In the absence of Irgm1, Irga6 co-localizes with lysosomes. a Wild type (WT), Irgm1 ^−/−, Irgm3 ^−/−, and Irgm1/Irgm3 ^−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irga6 antiserum (165/3) and anti-LAMP1 antibody. Representative microscopic images of Irga6 and lysosome co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 2a, showing percentage of Irga6 aggregate-like structures co-localizing with LAMP1. Irga6 does not form aggregate-like structures in WT cells and therefore it was not quantified; 100 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Gene switch (gs) 3T3 cells stably transfected with inducible Irga6 were stimulated with mifepristone and simultaneously transiently transfected with pGW1H-Irgm1, pGW1H-Irgm2, and pGW1H-Irgm3 either alone or in combination and incubated for 24 hours. Samples were fixed and stained as in 1a. Representative images of Irga6 and lysosome co-localization are shown. Images of the cells transfected with additional combinations of GMS proteins are also included (Additional file 2: Figure S2). Arrows point at Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: overlay, lower right: phase contrast. Scale bars represent 10 μm. d Quantification of 2c and S2, showing percent of Irga6 structures co-localizing with LAMP1; 50 cells per sample were quantified and counts of two independent experiments are shown. e Irgm1 ^−/− MEFs were induced with 200 U/mL IFN-γ and simultaneously transiently transfected with pEGFP-Irga6-ctag. Upon 24 h cells were fixed and stained for LAMP1. Scale bars represent 10 μM