Fig. 4: Function of SLP76 in RAGE-mediated activation of proinflammatory signaling pathways.

a Effects of RAGE and SLP76 overexpression on proinflammatory signaling pathways in HEK293 cells treated with AGEs. Flag-tagged SLP76 and HA-tagged RAGE were overexpressed together or separately in HEK293 cells treated with or without AGEs (100 μg/ml) for 15 min. Plasmids expressing a Flag or HA tag were used as controls. Phosphorylation of MAPKs and IKKα/β was detected by western blotting (n = 3). b–d Quantification of phosphorylated protein kinases activated by treatment with AGEs. The levels of phosphorylated p38 MAPK (b), ERK1/2 (c), and IKKα/β (d) were quantified with ImageJ software. Data were from three independent experiments (n = 3) and are shown as the mean ± SD. **P < 0.01, ****P < 0.0001 versus the group transfected with SLP76 alone, ^####P < 0.0001 versus cells transfected with RAGE alone (one-way ANOVA plus Tukey post hoc). e Effects of RAGE and SLP76 on AGE-induced activation of p38, ERK1/2, and IKKα/β in RAW264.7 cells. After transfection with control siRNA or with specific siRNA targeting AGER or SLP76 for 48 h, RAW264.7 cells were stimulated with AGEs (100 μg/ml) for 15 min (n = 3). f BMDMs isolated from WT, AGER knockout (AGER^−/−), and SLP76 knockout (SLP76^−/−) mice were treated with AGEs (100 μg/ml) for 15 min. Cells were harvested for detection of p38 MAPK, ERK1/2, and IKKα/β phosphorylation. The results represent three independent experiments (n = 3). g Effect of the intracellular region of RAGE on AGE-induced activation of signaling pathways. Flag-tagged full-length SLP76 and HA-tagged WT RAGE or RAGE deletion mutants were overexpressed in HEK293 cells. After treatment with AGEs (100 μg/ml) for 15 min, cells were harvested to detect phosphorylation of p38 MAPK, ERK1/2, and IKKα/β (n = 3). h Role of the SLP76 domain interacting with RAGE in AGE-induced activation of p38 MAPK, ERK1/2, and IKKα/β. HEK293 cells were cotransfected with plasmids expressing HA-tagged RAGE and Flag-tagged WT SLP76 or mutant SLP76. After transfection for 24 h, cells were treated as indicated. Western blotting was performed to detect phosphorylation of p38 MAPK, ERK1/2, and IKKα/β (n = 3). Source data are provided as a Source data file.