FIGURE 4.

Effects of TNF-α treatment on GKAP42 protein level. A, after being serum-starved for 3 h, 3T3-L1 adipocytes were treated with 2 nm TNF-α for the indicated periods. Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. Bands were quantified from each blot by ImageJ software. Protein amount of GKAP42 was calculated, and values are shown in the graphs. The results are presented as the means ± S.E. (error bars) of triplicate determinations. *, the difference versus 0 h of TNF-α treatment is significant, with p < 0.05. B, total RNA was extracted from 3T3-L1 adipocytes. Using this total RNA, first strand cDNA was synthesized, and PCR was carried out using these first strand cDNA as templates. Shown are representative data of independent triplicate determinations. C, 3T3-L1 adipocytes were serum-starved for 3 h and treated with MG-132, and cells were then treated with 2 nm TNF-α for 24 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Bands were quantified from each blot by ImageJ software. The protein amount of GKAP42 was calculated, and values are shown in the graphs. The results are presented as the means ± S.E. of three different experiments. There are significant differences between values with different letters (p < 0.05). D, 3T3-L1 adipocytes were infected with recombinant lentivirus expressing GFP or GFP-GKAP42. Then cells were pretreated with or without 2 nm TNF-α for 24 h and treated with or without 100 nm insulin for 5 min. Cell lysates were immunoprecipitated with anti-IRS-1 antibody, and immunoprecipitates (IP) or total cell lysates (TCL) were immunoblotted with the indicated antibodies. Bands were quantified from each blot by ImageJ software. Protein amounts of phosphorylated IRS-1 and phosphorylated Akt were calculated, and values are shown in the graphs. The results are presented as the means ± S.E. of three different experiments. *, the difference between GFP and GFP-GKAP42 expression with TNF-α treatment is significant, with p < 0.05.