Fig. 4.

Cbl triple-deficiency triggers stress response. (A) Stress and cell death-related gene expression was compared using the Mouse Stress & Toxicity PathwayFinder PCR Array between Cre adenovirus-infected WT and TMut MECs prepared as in Fig. 1A. RNA was isolated 24 h after infection. The solid line indicates equal expression. Dotted lines indicate 50% changes relative to WT. Raw data are available as Dataset S1. (B) qRT-PCR analysis of representative stress pathway transcripts in Cbl triple-deficient MECs. Expression levels of Ddit3, Hspa5, spliced and total Xbp1 and Hmox1 relative to Gapdh were determined. WT cells treated overnight with 200 nM thapsigargin (Thap) or 10 μM hydrogen peroxide (H₂O₂; Hmox1 only) were used as control. Levels in WT cells are set as 1. Data represent mean ± SEM of three independent experiments. (C) Immunoblot analysis of stress pathway molecules. For select molecules, lysates from L929 mouse cells treated with 1 μM thapsigargin for 6 h were used as a positive control (Thap). Phospho-IRE1α migrates faster than IRE1α (52).