Figure 3. Expression of the perilipin-1 439fs mutation in stably transduced 3T3-L1 pre-adipocyte cell lines.

3T3-L1 pre-adipocytes stably expressing either empty vector (EV) or N-terminally Myc tagged perilipin-1 wild type (WT), perilipin-1 439fs (439fs) and 398fs (398fs) mutants were generated as described in the materials and methods. A) Immunoblot analysis of perilipin-1 expression using an anti-Myc antibody, β-actin was used as a loading control. B) mRNA levels of hPLIN1 normalized to mCyclophilinA expression. C, D) Stably transfected 3T3-L1 pre-adipocytes expressing WT and 439fs perilipin-1 were grown to confluency and treated with 100 μg/ml cycloheximide (CHX) for the indicated times. Control (CT) cells were not treated with CHX. Cells were lysed and perilipin-1 expression levels were detected by immunoblotting with anti-Myc antibody and β-actin to assess loading. C) A representative image is shown. D) Quantification of perilipin-1 WT and 439fs expression levels from 3 independent experiments. Data represented as mean ±SEM. E) Stably transfected 3T3-L1 pre-adipocytes expressing WT and 439fs perilipin-1 were grown to confluency and treated with 100 μg/ml cycloheximide (CHX) and/or 10 μM MG132, or 25mM NH₄Cl for 24 hours. Control (CT) cells were not exposed to CHX. Cells were lysed and perilipin-1 and 2 expression levels detected by immunoblotting, β-actin was used to assess loading. A representative image is shown. *Non-specific band; arrow denotes corresponding band.