BFA, Tg and Tm differentially induce UPR sensors in the liver. (A,B) Standard PCR (A) and qPCR (B) analysis of xbp1 splicing in the livers of fish treated from 3 to 5 dpf with DMSO, 1 μg/ml BFA, 0.75 μM Tg or 1 μg/ml Tm using primers that amplify both unspliced (-u) and spliced (-s) xbp1. The average ratio of xbp1-second to xbp1-t is indicated (n=2). (C) Protein extracts from livers dissected from 5-dpf larvae treated as in A were blotted with anti-P-Eif2a and anti-tubulin as a loading control. Quantification and normalization to tubulin and DMSO controls is shown (n=2). (D) Analysis of atf4 and atf6 expression in livers of fish treated as in A. In B and D, target gene expression was normalized to rpp0 and fold changes compared to DMSO are plotted; n=13 for DMSO, 10 for Tm, 8 for Tg and 6 for BFA. Bars represent standard error in all graphs. *P<0.05, **P<0.01, calculated using paired Wilcoxon test.