Triglyceride Content Cells were incubated with 250M of IGOB131 for 72 h at 37 C in a humidified 5% CO2 incubator. Cells were col lected and lysed in lysis buffer. The total triglyceride content in cells was determined using a commercial triglyceride selleck kinase inhibitor assay kit. The protein concentration was determined by using a BioRad DC pro tein assay kit. Inhi bition was expressed as percent decrease in triglyceride content against control. Glycerol 3 Phosphate Dehydrogenase Activity 3T3 L1 adipocytes were harvested 8 days after the initia tion of differentiation and were incubated with 250M of IGOB131 for 72 h at 37 C in a humidified 5% CO2 incu bator. Cells were washed twice with ice cold PBS on 3T3 L1 adipocytes, and lysed in 25 mM Tris/1 mM EDTA, pH 7. 5 for the measurement of glycerol 3 phosphate dehy drogenase specific activity.
G3PDH activity was determined according to the procedure of Wise and Green. Protein concentration was determined by the Bio Rad DC protein assay kit using bovine serum albumin as a standard. Enzyme activity was expressed as units of activity/mg protein. Inhi bition was expressed as percent decrease in G3PDH activity against control. Western Blot Assay Cells were incubated with 0 250M of IGOB131 acids for 12 and 24 h at 37 C in a humidified 5% CO2 incuba tor. They were collected and lysed in ice cold lysis buffer, 2 mM EDTA, 500M sodium orthovanadate, 1% Triton X 100, 0. 1% SDS, 10 mM NaF, 10g/mL leupeptin and 1 mM PMSF. The protein con centration was estimated with the Bio Rad DC protein assay using bovine serum albumin as a standard.
Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 12% polyacryla mide gel. The proteins in the gel were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in PBST for 1 h. Membranes were incubated with primary anti body at 4 C overnight and then with secondary antibody for 1 h. Membranes were washed in PBST for 10 min three times between each step. The signal was detected using the Amersham ECL system. The relative expression of PPAR, adiponectin, and leptin in 3T3 L1 adipocytes was quanti fied densitometrically using the software LabWorks 4. 5, and calculated according to the reference bands of actin. Statistical analysis Values are expressed as mean S. E. For multiple compari sons, a one way analysis of variance was used.
When ANOVA showed significant differences, post hoc analysis was performed with the Newman Keuls multiple range test using SPSS. Results Effect of IGOB131 on the inhibition of Intracellular Triglycerides and G3PDH activity Carfilzomib in 3T3 l1 adipocytes The effect of IGOB131 on percent intracellular triglyceride and G3PDH levels were evaluated as indicated in the method section and the results are presented in Table 1. The reported values are the means SD of three samples. For this study cellular harvesting and incubation was accomplished with IGOB131 as previously described in the method section.