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. 2018 Oct 24;293(50):19365–19376. doi: 10.1074/jbc.RA118.004872

Figure 5.

Figure 5.

Up-regulation of XBP1s in CNP-deficient mouse ES cells. A, design of an sgRNA targeting an XbaI site in exon 2 of mouse CNP gene. Resulting clones were genotyped by PCR amplification of exon 2 and scanned by XbaI digestion. F, forward; R, reverse. B, a CNP−/− ES cell clone was identified with frameshift mutations in both alleles. Western blotting showed the loss of CNP protein. C, RT-qPCR analysis of spliced and total XBP1 mRNA levels in WT, CNP−/−, and genetically rescued CNP−/− ES cells. ER stress was induced with thapsigargin for 1, 2, and 3 h. D, CNP protein levels in the samples analyzed in C. Transfected hCNP was tagged with 3xFLAG. We observed that the translation was initiated from the start codons of both FLAG and CNP; thereby two bands were observed in the Western blot. E, expression of XBP1 target genes EDEM and Erdj4 were analyzed by RT-qPCR. Error bars represent S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.