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. 2009 Jan 12;29(6):1459–1471. doi: 10.1128/MCB.00754-08

FIG. 2.

FIG. 2.

The interaction between ClipR-59 and Akt. (a) Coimmunoprecipitation assay of endogenous ClipR-59 and Akt from 3T-L1 adipocytes. Differentiated 3T3-L1 adipocyte lysates were immunoprecipitated (IP) with anti-Akt antibodies (Upstate Biotechnology). The anti-Akt immunoprecipitates were analyzed in a Western blot assay (IB) with anti-ClipR-59 antibodies. Rat IgG served as the control. (b) Coimmunoprecipitation assay of endogenous ClipR-59 and Akt from differentiated 3T-L1 adipocytes. 3T3-L1 adipocyte lysates were immunoprecipitated with rabbit monoclonal antibodies to Akt1 and Akt2 (Cell Signaling). The anti-Akt immunoprecipitates were analyzed in Western blot assays with anti-ClipR-59 antibodies. Rat IgG served as the control. (c) Coimmunoprecipitation assay with HA-tagged Akt1 or Akt2 and Flag-tagged ClipR-59 proteins in transfected HEK293 cells. Top: Western blot of HA-tagged Akt immunoprecipitates, showing recovery of Flag-tagged ClipR-59. Middle: input of Flag-tagged ClipR-59. Bottom: input of HA-tagged Akt. WT1, wild-type Akt1; ΔPH, Akt1 without PH domain; WT2, wild-type Akt2. (d) Coimmunoprecipitation assay of HA-tagged Akt1 or Akt2 and Flag-tagged ClipR-59 proteins expressed in HEK293 cells with anti-Flag antibodies. (e) Confocal microscopy analysis of the cellular location of HA-Akt (red) and ClipR-59-GFP (green) expressed in COS-7 cells. The blue is the DAPI staining of nuclei. (f) Confocal microscopy analysis of the cellular location of endogenous Akt (red) and ClipR-59-GFP (green) in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were infected with ClipR-59-GFP-expressing adenovirus. At 36 h postinfection, the cells were fixed and stained with rabbit monoclonal Akt2 antibodies (red). The blue indicates the nuclei stained with DAPI.