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. 2023 Jul 21;14:1182180. doi: 10.3389/fimmu.2023.1182180

Figure 1.

Figure 1

Ii interacts with Rai14. (A) Lysates from MelJuSo cells were subjected to immunoprecipitation with an antibody against Ii (MB741) or an isotype control (IgG2ακ). Immunoprecipitated (IP) samples and whole cell lysates (WCL) were analyzed by western blot using antibodies against Ii and Rai14. (B) MelJuSo cells were subjected to immunoprecipitation with an antibody against Rai14 or an isotype control (IgG). Immunoprecipitated (IP) samples and whole cell lysates (WCL) were analyzed by western blot using antibodies against Ii and Rai14. (C) An antibody targeting Ii (M-B741) conjugated with Alexa fluor 555 was added to MelJuSo cells co-transfected with Ii p33 and Rai14-GFP before live cell imaging. Arrowheads in the magnifications of the boxed area point to membrane ruffles positive for both Ii and Rai14, while the arrows point to a Rai14 and Ii-positive membrane tubule from where an Ii-positive vesicle pinches off. Scale bars, 10 μm. (D) Rai14 colocalization with Ii over time for (C) was quantified using Image J as Mander’s colocalization coefficients. (E) MelJuSo cells co-transfected with Iip33, mCherry-Iip33, and Rai14-GFP. Arrowheads in the magnifications of the boxed area point to Rai14 domains present on Ii-positive endosomes. Scale bars, 10 μm. The graphs show the normalized fluorescence intensity profiles relative to Rai14 (green) and Ii (red) along the lines illustrated in the insets.