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. 2017 Mar 10;12(3):e0173264. doi: 10.1371/journal.pone.0173264

Fig 6. Effects of coffee extract on expression of IRβ, IRS1, and IRS2 in 3T3-L1 cells.

Fig 6

(A) 3T3-L1 cells were treated with 5% (v/v) coffee extract for 1 hr, and then stimulated with MDI for the indicated periods. Cell lysates were immunoprecipitated (IP) with an anti-IRS1 antibody, followed by immunoblotting (IB) with an anti-phospho-tyrosine (PY) antibody or anti-IRS1 antibody. Whole cell lysates were immunoblotted with an anti-β-actin antibody. The phosphorylation level of IRS1 was normalized with the expression level of IRS1 or the expression level of β-actin. The relative phosphorylation of IRS1 is shown in the graphs. Values are the mean ± S.D. of three independent experiments. **p<0.01 vs. control cells. (B, C) 3T3-L1 cells were treated with 5% (v/v) coffee extract for the indicated periods. (B) Cell lysates were immunoblotted with an anti-IRβ antibody, anti-IRS1 antibody, anti-IRS2 antibody, or anti-β-actin antibody. The expression level of IRβ, IRS1, and IRS2 was normalized with the protein amount of β-actin. The relative expression level of IRβ, IRS1, and IRS2 is shown in the graphs. Values are the mean ± S.D. of three independent experiments. (C) Total RNA was prepared and the mRNA expression of Irβ, Irs1, and Irs2 was analyzed using quantitative real-time PCR. β-actin mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. **p<0.01 vs. untreated cells.