Figure 4. ER stress increases Derlin-1 expression and promotes the recruitment of TER94 to the ER.

(A) Confocal images of control GMR>LacZ, GMR>TER94^K2A, and derlin-1^null larval eye discs expressing CD3δ-YFP (green). The eye discs are stained with anti-Elav antibodies (red) to label neuronal nuclei. Expression of TER94^K2A serves as a positive control for CD3δ-YFP. The boxed region in the derlin-1^null panel is shown at a higher magnification. (B) Quantitative RT-PCR analysis of derlin-1 and bip transcripts from eye discs with (+) and without (−) 5 mM DTT treatment. Results from three independent quantitative RT-PCR experiments, after being normalized to rp49 levels, are shown in fold change (compared to untreated). Values shown represent mean ± SE. *p<0.05; **p<0.01 (Student's t-test). (C) Confocal images of wild-type mid-pupal eyes with and without (−) 5 mM DTT treatment, stained with phalloidin (red) and anti-Derlin-1 (green). (D) Quantitative Western of endogenous Derlin-1 protein levels from flies subjected to 2 hrs cold shock at 0°C. Lysates from wild-type flies (con) and those recovered after the cold shock for the indicated time periods are probed with anti-Derlin-1 antibody. β-Tubulin levels serve as loading control. (E) Results from eight independent experiments in D are shown. Derlin-1 protein levels, normalized to loading controls, are shown in fold change as compared to untreated control. Values shown represent mean ± SE. *p<0.05; **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test). (F and G) Confocal images of GMR>KDEL-eGFP mid-pupal eyes, before (“−“ in F; left panels in G) and after the cold treatment (cold shock), stained with anti-Derlin-1 (F) or anti-VCP (G) antibodies (red). KDEL-eGFP (green) labels the ER, and the co-localization with KDEL-eGFP in merged panels is shown in white. (H) Pearson's co-localization coefficient analyses of images from four independent experiments as in G (see Materials and Methods for details). Cold shock treatment shows enhanced correlation of pixel pairs that label TER94 and the ER in a Derlin-1-dependent manner. Scale bars: 10 µm. (I) Western analysis of lysates and anti-VCP immunoprecipitates from flies treated with (4 hrs) or without (con) cold shock. The IP blot was probed with anti-VCP and anti-Derlin-1 to detect TER94/Derlin-1 complexes. The lysate (input) blot was detected by anti-VCP, and then stripped and re-probed with anti-β-Tubulin for loading control.