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. Author manuscript; available in PMC: 2011 Feb 5.
Published in final edited form as: Cell. 2010 Feb 5;140(3):338. doi: 10.1016/j.cell.2010.01.001

Figure 3. PKR directly regulates IRS-1 phosphorylation.

Figure 3

(A) Induction of interaction between IRS-1 and PKR after TNFα treatment in Pkr+/+ and Pkr−/− MEFs. IRS-1 and PKR protein level were examined either with immunoprecipitation (IP) followed by immunoblotting (IB) or by direct immunobloting in cells treated with 5 ng/ml TNFα treatment for 3 hours.

(B) Physical interaction between IRS-1 and PKR in TNFα-treated MEF cells. Cell lysates were prepared from Pkr+/+ or Pkr−/− MEFs treated with 5 ng/ml TNFα for 3 hour followed by immunoprecipitation with anti-PKR antibody and western blot analysis with anti-IRS-1 antibody.

(C) Physical interaction between IRS-1 and PKR in a pull-down assay in vitro using recombinant IRS-1 and PKR proteins.

(D) Direct phosphorylation of IRS-1 by PKR in kinase assay in vitro using recombinant IRS-1 and PKR proteins. Phosphorylation level of IRS-1 was assessed by autoradiography or western blot analysis with anti-phospho-IRS-1 (serine 307) antibody.

(E and F) In vitro PKR kinase assay. IRS-1 phosphorylation by immunopurified PKR prepared from 5 ng/ml TNFα (E)- or 300 nM thapsigargin (F)-treated MEFs and analyzed by autoradiography or western blot analysis with anti-phospho-IRS-1 (serine 307) antibody.

(G) Effects of exogenous expression of PKR on IRS-1 serine phosphorylation in primary Jnk1+/+ and Jnk1−/− MEFs. Flag-tagged human PKR was introduced to primary Jnk1+/+ and Jnk1−/− MEFs by adenovirus-mediated gene transfer. Phosphorylation level of IRS-1 on serine 307 was examined with anti-phospho-IRS-1 (serine 307) antibody. Both exogenous and endogenous PKR expression was detected by anti-PKR antibody. The graph on the right shows the quantification of the data.

See also Supplemental Figure S3.