Semi-Quantitative PCR Analysis Of CHOP Exon

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Figure 5: Semi-quantitative PCR analysis of CHOP splicing variants in plasmids and liver tissues, and CHOP exon arrangements. (A) CHOP1 mRNA contains four exons, whereas CHOP2 mRNA lacks the second exon of CHOP1. The 5 '-leader of CHOP is encoded within the first and second exons, while the coding sequence is encoded within the third and fourth exons of CHOP. (B) As expected, CHOP1 primers amplified CHOP1 transcripts at the size of 214 bp in the CHOP1 plasmid template containing the full sequence of CHOP1. While, CHOP2 isoforms were detected at the size of 207 bp in the CHOP2 plasmid expressing the whole sequence of CHOP2. The presence of a single band amplified by the corresponding primers in each plasmid indicates the specificity of CHOP primers. (C) CHOP1 and CHOP2 are also expressed in tunicamycin-treated liver tissues at the expected sizes 214 bp and 207 bp, respectively. Also, β-actin was used as a control. The common exons are represented as black boxes, and the alternative exon is shown as a white box. Introns are illustrated as lines connecting exons. P1, primers specific for CHOP1 detection; P2, primers specific for CHOP2 detection; NT, non-treated liver tissues; Tm/6hr, liver tissues treated with tunicamycin for 6 hours. …show more content…
(A) The CHOP1-luc plasmid is constructed to have an uORF preceding the luc ORF to determine the effect of CHOP1 uORF on luciferase activity. The CHOP2-luc plasmid is constructed to have CHOP2 uORF that overlaps out-of-frame with the luciferase CDS. (B) In comparison to non-treated (NT) MEF cells, CHOP1 and CHOP2 showed nearly a 2.5-fold induction of luciferase activity in MEF cells treated with thapsigargin (Tg), an ER stress agent, for 12 hours (12 hr). These findings indicate that CHOP1 and CHOP2 are preferentially translated during ER

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