Figure 1. Rab11 FIP proteins regulate adiponectin release in HEK293 cells.

A) GFP tagged wild type RAB11-FIPs and myc tagged adiponectin were transiently co-transfected in HEK293 cells. 24hr following transfection adiponectin was quantitated by ELISA as indicated in the materials and methods section B) Knock down of FIP proteins in HEK293 cells. HEK293 were left untransfected or transfected with a shRNA empty vector (pKLO.1puro) or transfected with a pool of plasmid expressing shRNA sequences specific for FIP proteins as indicated. Whole cell lysates were obtained and protein samples were separated on SDS-PAGE, transferred to nitrocellulose filters and immunoblotted with specific antibodies detecting the human isoforms of FIP1, FIP3 and FIP5 proteins. Anti β-tubulin antibody was used as loading control. C) Quantification of the FIP knockdown efficiency for each FIP protein. Data are mean ± SEM from three independent experiments. D) HEK293 were transfected with a shRNA expression (pKLO.1puro) empty vector or shRNA vectors containing shRNA sequences for FIP proteins (as indicated, mission shRNA SIGMA). Cells were selected for 96 hours in puromycin (2 µg/ml) containing media and surviving cells were transfected with a plasmid coding for adiponectin-myc. 24hr following transfection adiponectin was quantitated by ELISA in cell lysates and media and adiponectin secretion was calculated as indicated in the materials and methods section. Data are means ± SEM of at least three independent experiments Statistical analysis: One way ANOVA * indicates statistically different from control cells (p < 0.05).