Figure 4
From: Enhanced expression of Survivin has distinct roles in adipocyte homeostasis
Ectopic expression of Survivin inhibits lipolysis by targeting Adrb3 and Fsp27. (a) 3T3-L1 adipocytes were stimulated with 1 μM isoproterenol (ISO) for 3 h, then media was removed for glycerol measurement. (b) 3T3-L1 adipocytes were stimulated with 1 μM ISO for 1 h and also treated with 200 μM IBMX along with ISO stimulation, then cells were extracted for cAMP assay. (c) 3T3-L1 adipocytes were stimulated with 1 μM ISO for indicated time. Total proteins were subjected to SDS-PAGE. Immunoblots for GAPDH, phospho-HSL at Ser-563, Ser-565, and Ser-660, phospho-perilipin using phospho (Ser/Thr)-PKA substrate antibody to detect protein at 62 kDa and total HSL and perilipin were performed on cell lysates. (d) Fsp27 stability was evaluated in a cycloheximide (CHX, 100 μg/ml) chasing experiment. Immunoblot of Fsp27 after 15, 30 and 60 min treatments with CHX in 3T3-L1 adipocytes. ‘Black circles’=control group; ‘black squares’=Survivin overexpression group. (e) 293T cells were infected with Birc5 lentivirus to overexpress Survivin, then transfected with flag-tagged Fsp27, as indicated, and the lysates were immunoprecipitated with flag beads. *P<0.05, **P<0.01, ***P<0.001
