Extended Data Fig. 8: SCG2-LILRB4 interaction upregulates IL-6-STAT3 activation.
From: Secretogranin 2 binds LILRB4 resulting in immunosuppression
a, Quantification and statistical analysis of Western blot results shown in Fig. 6a (n = 3 biological replicates). b-c, Immunoblot (b) and quantification (c) of p-STAT3 levels in BM-MDSCs from LILRB4-Tg mice stimulated with or without IL-6 for 10, 20, 30 min in plates coated with BSA, SCG2 or ApoE (n = 3 biological replicates). d-e, Immunoblot (d) and quantification (e) of p-STAT3 levels in BM-MDSCs from WT, LILRB4-Tg, or LILRB4-Tg ApoE−/- mice. Cells were stimulated with or without IL-6 for 10 min in plates coated with BSA, SCG2 or ApoE (n = 3 biological replicates). f, Quantification and statistical analysis of Western blot results shown in Fig. 6b (n = 3 biological replicates). g, mRNA level of Arg1 in BM-MDSCs stimulated with IL-6 under different conditions. Cells were incubated with varying concentration of IL-6 for 24 h or with IL-6 for indicated times in plates coated with BSA or SCG2 (n = 3 biological replicates). h, IP Flag-tagged LILRB4 and endogenous STAT3 in THP-1 cells. THP-1 LILRB4-Flag stable cells were subjected to IP with anti-Flag beads followed by IB with anti-STAT3 antibody. i,Co-IP of Flag-tagged STAT3 domain deletion and HA-tagged LILRB4 in HEK293T cells. j, SCG2 induced phosphorylation of STAT3 in a recombinant HEK293T system. HEK293T cells were co-transfected with LILRB4-Flag, ITIM-mutant LILRB4-Flag (2 μg), STAT3 (1 μg), Lyn (100 ng), and SCG2 (300 ng) as indicated. At 48 h post-transfection, cell lysates were collected and subjected to IP/IB analysis. k, Quantification and statistical analysis of Western blot results shown in Fig. 6j (n = 3 biological replicates). l, Immunoblot of p-STAT1 and p-STAT6 in BM-MDSCs from LILRB4-Tg mice stimulated with or without IFN-γ or IL-4 (20 ng/mL) for 10 min in plates coated with BSA or SCG2. m, Quantification and statistical analysis of Western blot results shown in Fig. 6l (n = 3 biological replicates). Data are presented as mean ± s.d. p values were determined by one-way ANOVA with Tukey’s multiple comparisons test (a, f, k, and m), two-way ANOVA with Tukey’s multiple comparisons test (c and e) or two-way ANOVA with Bonferroni’s multiple comparisons test (g).
