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. Author manuscript; available in PMC: 2021 Dec 28.
Published in final edited form as: Cell Rep. 2020 Jun 30;31(13):107781. doi: 10.1016/j.celrep.2020.107781

Figure 1. CARMIL1 Associates with IL-1 Signaling Proteins to Mediate ERK Phosphorylation.

Figure 1.

(A) Whole-cell lysates and BSA- and collagen-coated bead-associated proteins were prepared from human gingival fibroblasts (HGFs) that had been treated without or with IL-1 (20 ng/mL) for indicated time periods. Lysates were immunoblotted for CARMIL1, paxillin, and GAPDH. Densitometry data are expressed as means ± SE. Differences between indicated groups were assessed by ANOVA.

(B) CARMIL1 and IgG (top) immunoprecipitates (IPs) were prepared from whole-cell lysates of HGFs that had been treated without or with IL-1 (20 ng/mL) for 45 min and immunoblotted for IL-1R1, IRAK, CARMIL1, and IgG, respectively. Densitometry data of the ratios of IL-1R1/CARMIL1 and IRAK/CARMIL1 are expressed as means ± SE. IL-1R1 and IgG (bottom left) and IRAK and IgG (bottom right) IPs were prepared from whole-cell lysates of HGFs that had been treated without or with IL-1 (20 ng/mL) for 45 min and immunoblotted for CARMIL1, IL-1R1, IRAK, and IgG, respectively. Densitometry data of ratio of CARMIL1/IL-1R1 and CARMIL1/IRAK are expressed as means ± SE.

(C) HGFs were transfected with siRNA control or siRNA CARMIL1 for 48 h and then treated with either PBS or IL-1 (20 ng/mL) for 45 min. Lysates were immunoblotted for p(T202/Y204)-ERK and total ERK. Lysates of siRNA control and siRNA CARMIL1-transfected cells were immunoblotted for CARMIL1 to confirm CARMIL1 KD. β-actin was used for loading control. Ratios of p(T202/Y204)-ERK to ERK were quantified by densitometry, which were assessed by ANOVA and are expressed as means ± SE.