Supplemental Data


Abstract
Full Text
Reversal of Obesity- and Diet-Induced Insulin Resistance with Salicylates or Targeted Disruption of Ikkbeta
Minsheng Yuan, Nicky Konstantopoulos, Jongsoon Lee, Lone Hansen, Zhi-Wei Li, Michael Karin, and Steven E. Shoelson

Supplementary Material

Supplemental Figure 1. Electrophoretic mobility shift of IRS-1. (A) IRS-1 was immunoprecipitated from liver homogenates and identified by blotting with antibodies to IRS-1. IRS-1 from control animals consistently exhibited slower electrophoretic mobilities as compared with IRS-1 from animals treated with aspirin or sodium salicylate. (B) Electrophoretic mobilities increased and were identical after treatment with alkaline phosphotase (AP).


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Supplemental Figure 2. Induction of insulin resistance with I(B kinases and reversal with dominant inhibitors. (A to C) Myc-tagged NIK, Flag-tagged IKKalpha, and Flag-tagged IKKbeta, or (D to F) kinase-deficient versions of IKKalpha, and IKKbeta, 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 mug 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-alpha followed by 5 min stimulations with 10 nM insulin. Protein expression levels ranged from 10 to 20 times that of the endogenous proteins.


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