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. 2009 Dec 10;59(3):714–725. doi: 10.2337/db09-0911

FIG. 9.

FIG. 9.

Regulation of signaling via Hsf-1, Akt, JNK, and FOXO-1 by RLIP76. RLIP76+/+ and RLIP76−/− MEFs were subjected to transfection with pcDNA3.1 empty vector (V) or pcDNA3.1 with full-length RLIP76 cDNA (R) using Lipofectamine (Invitrogen). Crude supernatant of 28,000g of these cells was subjected to Western blot analysis against anti-RLIP76 IgG to demonstrate the absence of RLIP76 antigen in RLIP76−/− MEFs and to show increased RLIP76 protein upon transfection in both RLIP76+/+ and RLIP76−/− MEFs. β-Actin was used as an internal control (A). The effect of RLIP76 overexpression, insulin, or anti-RLIP76 antibody on RLIP76+/+ and RLIP76−/− MEFs on signaling as well as glucose uptake was carried out at 37°C with 5% CO2 atmosphere. The cells were pretreated with either polyclonal rabbit preimmune IgG (C or PIS) or anti-human RLIP76 IgG fractions (AR) (40 μg/ml final concentration) for 1 h. Buffer containing 14C-glucose and either no insulin or 20 mU insulin was added to start the measurement of glucose uptake, and the measurement was terminated at 30 min by washing off the medium with ice-cold PBS and solubilization of cells in counting cocktail. Glucose uptake was measured in 5 × 106 cells, as described previously (36). In parallel experiments in which 14C glucose was omitted, measurements of phosphorylation of Akt, Hsf-1, and JNK, and inactivation of Foxo1, were performed at 30 min. Results of Western blot analyses of Hsf-1 expression and phospho-Akt (Ser473; Millipore, Billerica, MA) are presented. β-actin expression was shown to confirm equal amount of protein was loaded in each sample (B). Results from all groups were analyzed with plots of glucose uptake vs. Hsf-1 expression and Akt activation as well as Foxo-1 inactivation and JNK phosphorylation (measured by ELISA; Active Motif, Carlsbad, CA) (C). Results of all five measurements normalized to the control group (RLIP76+/+ MEFs transfected with empty vector, treated with no insulin and preimmune IgG) are presented (D). Means ± SD for two separate experiments, each in triplicate, are shown; n = 6 (33,36).

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