Figure 3.

Endogenous lipin 1 inducibly binds to seipin in maturing adipocytes. (A) Following differentiation for 7 days control 3T3-L1 adipocytes or adipocytes stably expressing myc-sepin were serum starved for 2 h. Adipocytes were left untreated (b) or incubated with 330 μM each of oleate and linoleate (F), 100 nM insulin (I) or a combination of both (FI) for 30 min. Thirty micro grams of cell lysates or anti-myc immunoprecipitates were western blotted for myc-seipin, endogenous lipin 1 or calnexin. (B) Myc-seipin expressing adipocytes were incubated for 30 min in the absence (b) or presence (FI) of 330 μM each of oleate and linoleate and 100 nM insulin, fixed and immunostained for myc-seipin and endogenous lipin 1. Individual images are shown in grayscale and an ROI containing peak myc-seipin staining of each cell shown at higher magnification (inset). Merged image shows overlay of myc-seipin (green) and lipin 1 (red). White arrows reference regions of high seipin intensity. (C) Quantification of endogenous lipin and myc-seipin co-localisation in adipocytes under basal (Bas) conditions or following treatment with 330 μM each of oleate and linoleate and 100 nM insulin for 30 min (FI) n=12. Data shown are ±SEM, * indicates difference from basal p<0.005. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)