Journal: Cell metabolism
Article Title: Autonomous sensing of the insulin peptide by an olfactory G protein-coupled receptor modulates glucose metabolism.
doi: 10.1016/j.cmet.2021.12.022
Figure Lengend Snippet: Figure 5. InsB:9-23 induced CCL2 production by activating the b-arrestin-1-SRC-STAT3 pathway (A) CCL2 secretion from islets isolated from WT mice pre-treated with or without PTX or from Arrb1–/– mice in response to 100 nM insB:9-23 stimulation (n = 6). (B–D) Representative blots (B) and quantification (C and D) of the protein and phosphorylation levels of SRC, STAT3, and NFkB P65 subunit in control or b-arrestin-1 siRNA-transfected MIN6 cells treated with 100 nM insB:9-23 or control vehicle (n = 3). See also Figure S6Z. (E) Representative blots showing the coimmunoprecipitation of HA-b-arrestin-1 with Flag-b2AR and SRC or with Flag-OLFR109 and SRC in HEK293 cells. (F and G) Quantification of SRC (F) and receptor levels (G) after coimmunoprecipitation by HA antibody-conjugated agarose from the lysates of HEK293 cells. The data are correlated with (E) (n = 3). (H) Colocalization of b-arrestin-1 and SRC in HEK293 cells transfected with OLFR109, RFP-b-arrestin-1, and SRC-YFP in response to 100 nM insB:9-23 stimulation for 5 min. HEK293 cells transfected only with RFP-b-arrestin-1 and SRC-YFP were used as the control. Scale bar, 20 mm. (I) Pearson’s correlation analysis of RFP-b-arrestin-1 and SRC-YFP fluorescence intensities. The Pearson’s correlation coefficient was 0.62. (J) Effects of pretreatment with inhibitors of SRC, STAT3, or NFkB on insB:9-23-induced CCL2 secretion in the pancreatic islet isolated from WT mice using an ELISA kit (n = 6). ***p < 0.001; ns, no significant difference (A, C, D, F, G, and J). ###p < 0.001; ns, no significant difference (J). The bars indicate the mean ± SEM values. All data were statistically analyzed using two-way ANOVA with Dunnett’s post hoc test.
Article Snippet: The supernatants were collected and neutralized with 1M NaOH and the cAMP contents were determined using cAMP ELISA Kit (R&D systems, Cat# KGE012B) according to manufacturer’s instructions.
Techniques: Isolation, Phospho-proteomics, Control, Transfection, Enzyme-linked Immunosorbent Assay
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