|Reversal of Obesity- and Diet-Induced Insulin Resistance with Salicylates or Targeted Disruption of Ikk|
Minsheng Yuan, Nicky Konstantopoulos, Jongsoon Lee, Lone Hansen, Zhi-Wei Li, Michael Karin, and Steven E. Shoelson
Supplemental Figure 2. Induction of insulin resistance with I(B kinases and reversal with dominant inhibitors. (A to C) Myc-tagged NIK, Flag-tagged IKK, and Flag-tagged IKK, or (D to F) kinase-deficient versions of IKK, and IKK, were expressed in HEK 293 cells. The pRK7 (CMV) vectors encoding the kinases were obtained from M. Rothe (Tularik); transfections of 50 to 60% confluent cells (1.5 g DNA per well; six well dishes) with FuGene 6 (Boehringer-Mannheim) were as recommended. (A to F) Experiments were initiated 36 hours after transfection and 16 hours after the removal of serum from the culture medium. (A to C) Cells were stimulated for 5 min with 10 nM insulin unless otherwise indicated. (D to F) Cells were treated for 40 min with 6 nM mTNF- followed by 5 min stimulations with 10 nM insulin. Protein expression levels ranged from 10 to 20 times that of the endogenous proteins.