Fig. 4.

GDM and insulin effect on hCAT-1 expression in A₁AR and A_2AAR knockdown cells. a Western blot for hCAT-1 protein abundance in HUVECs in the absence (−) or presence (+) of insulin (1 nM, 8 h) in non-transfected (−) or transfected (+) cells with siRNA against A₁AR (^KDA₁AR). Lower panel, hCAT-1/β-actin ratio densitometries normalized to 1 in cells transfected with sc-siRNA from normal or GDM pregnancies in the absence of insulin. b Western blot for hCAT-1 protein abundance with siRNA against A_2AAR (^KDA_2AAR) as in A. c hCAT-1 mRNA expression in ^KDA₁AR or ^KDA_2AAR cells as in A. d Luciferase (Luc) reporter construct pGL3-hCAT-1⁻¹⁶⁰⁶ of SLC7A1 promoter transfected in ^KDA₁AR or ^KDA_2AAR cells, along with Renilla reporter plasmid. After 36 h of transfection, cells were incubated without (−) or with (+) insulin (1 nM, 8 h) (see “Materials and methods”). e Luciferase (Luc) reporter construct SLC7A1 promoter transfected in ^KDA₁AR or ^KDA_2AAR cells as in d. In a, *P < 0.05 vs. all other values except between themselves, †P < 0.05 vs. all other corresponding values in GDM. In b, *P < 0.05 vs. all other values. †P < 0.05 vs. all other values except between themselves. In c–e, *P < 0.05 vs. all other values in Normal except between themselves. †P < 0.05 vs. all other values in GDM except between themselves. ‡P < 0.05 vs. corresponding values in Normal. Values are mean ± SEM (n = 29)