Kinase suppressor of Ras-1 protects intestinal epithelium from cytokine-mediated apoptosis during inflammation
J. Clin. Invest. Fang Yan, et al. 114:1272 doi:10.1172/JCI21022 [
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Figure 4KSR1 is activated in inflamed colon mucosa. (
A) H&E staining of paraffin-embedded colon tissues prepared from
IL-10+/+ (BALB/c) or
IL-10–/– mice before developing overt signs of colitis (8-week-old mice) or with rectal prolapse (12-week-old mice) showing characteristic epithelial ulceration (magnification, ×20). (
B) Colon mucosal lysates were prepared from the respective mice for Western blot analysis with indicated antibodies. KSR1 (
C,
E, and
F) or Raf-1 (
D) was immunoprecipitated from colon mucosal lysates using anti-KSR1 or anti–Raf-1 antibodies, respectively, and subjected to Western blot analysis to detect phosphorylation levels using anti–P-Thr or anti–P-Ser antibodies. Using a 2-stage in vitro kinase assay (
E and
F), immunoprecipitated KSR1 was incubated first with recombinant Raf-1, and then Raf-1 was incubated with KI MEK1 as substrate as detailed in Methods. Phosphorylation of Raf-1 or MEK1 was determined using anti–P-Thr or anti–P-MEK1/2 antibodies, respectively. Immunoprecipitated KSR1 was treated with calf intestinal alkaline phosphatase (PTase) prior to incubation with Raf-1 (
F), as indicated. The results shown here are from 2 representatives of 5 mice. WBA, Western blot analysis.
