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. 2021 Sep 8;62(12):3. doi: 10.1167/iovs.62.12.3

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Figure 8. — Assessment of Xbp1 mRNA splicing in Epha2-knockout mouse lenses. Partial Xbp1 mRNA flanking the 26-nucleotide long intron was amplified from lenses (n = 4) of 8-week-old Epha2^+/+, Epha2^+/⁻, and Epha2⁻^/⁻ (A), and 14 to 19-week-old Epha2^+/+ and Epha2^+/⁻ (B), mice on C57BL/6J background by RT-PCR using gene-specific primers. The PCR product was digested with PstI restriction enzyme and size separated on a 3% agarose gel. In B, each lane represents analysis of lenses of two mice of the same sex. PstI digestion of the unspliced variant (uXbp1) at the PstI site in the intron resulted in 301 bp (u1) and 193 bp (u2) fragments. The spliced variant (sXbp1) generated by excision of the intron, as expected, was not digested by PstI. Both unspliced and spliced variants were detected in lenses of all genotypes and at both the ages, A and B. Asterisks marks a hybrid of the two variants. The proportion of the spliced variant in a sample was calculated (sXbp1/(uXbp1+sXbp1+u1+u2)). Mean percent spliced variant in lenses of each genotype is indicated; SD = standard deviation. The data in A was statistically analyzed by ANOVA with Dunnett's T test for multiple comparisons. ***p < 0.001; ^ versus Epha2^+/+.