Fig 2: The novel anti-IRAP antibodies can specifically detect human IRAP. (a) Representative Western blots (uncropped 12 lanes in same gel) showing binding of the novel mouse anti-IRAP antibodies (RB9, RF7, RH3, RG4; 0.5 µg/µl) to (1) full length IRAP derived from membrane preparations of HEK293T cells transiently overexpressing human IRAP (0.01 µg protein/µl) or (2) purified soluble human IRAP (0.001 µg/µl). The lanes for each antibody were imaged at either different exposure times or at the same exposure time across all antibodies (n = 3). Original, uncropped blots are provided in Supplementary Fig. 1. (b) The mouse anti-IRAP antibodies (1 µg/well) and the commercially available rabbit anti-IRAP antibody (Rb; 1:500 concentration) were tested for binding to mIRAP (10 µg protein/well) or purified sIRAP (0.001 µg/well) using an indirect ELISA. Data presented is the mean from two separate experiments performed in duplicate (n = 2). (c) The mouse RB9 clone (1 µg/well; red) and the rabbit antibody (1:500; purple) were tested against decreasing concentrations of purified sIRAP (0.001–0.01 µg/well) and mIRAP (10 µg protein/well). Simple linear regression was conducted (R2 = 0.94). Data presented is the mean of absorbance readings from one experiment performed in duplicate (n = 1).

Fig 3: TLR4 is highly expressed at the cell surface in male IRAP-deficient macrophages. (A) Representative immunofluorescent images of TLR4 (green) in untreated control (C) and LPS-treated (L) CD45+ (red) BMDM derived from male and female wildtype (WT) and IRAP knockout (KO) mice. (B) Semi-quantification of TLR4 expression from immunofluorescent (IF) images of BMDM measured as % area positive staining relative to the cell count. (C) Semi-quantification of the TLR4 expressed at the cell surface of BMDM in IF images measured as % colocalization of TLR4 and CD45. (D) Raw mean fluorescence intensity (MFI) of cell surface TLR4 expression in CD45+ F4/80+ CD206- M1 macrophages in the spleens of mice treated for 4 hours with vehicle control (V) or LPS (L) measured via flow cytometry. All data was analyzed using three-way ANOVAs with Sidak’s post-hoc test, **p<0.01, n=3. All data is presented as mean ± SEM.

Fig 4: IRAP gene deletion does not significantly alter macrophage activation in female BMDM. (A) Representative flow cytometry dot plots showing control (C) and LPS-treated (L) CD45+ F4/80+ CD206- M1 macrophages from female wildtype (WT) mice and (B) the frequencies (%) of the M1 macrophage population in WT (filled bars) and IRAP knockout (KO; empty bars) BMDM cultures. (C) Frequency curves of the activation markers CD40 and CD80 in C (blue) and L (red) WT (filled curve) and IRAP KO (empty curve) M1 macrophages. (D) Quantification of the fold changes in mean fluorescence intensity (MFI) compared to the mean of the controls of CD40, CD80, CD86 and MHCII in M1 macrophages. Data was analyzed using a two-way ANOVA with Tukey’s post-hoc test, ****p<0.0001 control vs LPS, ##p<0.01, ###p<0.001 WT vs KO, n=3. All data is presented as mean ± SEM.

Fig 5: IRAP-deficient BMDM have heightened LPS-induced increases in TNF-α and IL-1β expression. Representative immunofluorescent staining of (A) TNF-α or (B) IL-1β (green) in control (C) or LPS-treated (L) CD45+ (red) BMDM from male and female wildtype (WT) and IRAP knockout (KO) mice. (C) Protein concentrations (pg/ml) of TNF-α, IL-1β and IL-6 in the conditioned media of BMDM measured using a sandwich ELISA. Data was analyzed using three-way ANOVAs with Tukey’s post-hoc test, **p<0.01, ***p<0.001, n=5-6. Data is presented as mean ± SEM.