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. 2022 Mar 7;14(4):e14817. doi: 10.15252/emmm.202114817

Figure 4. Lack of harmonin does not alter expression of other USH proteins.

Figure 4

  1. RT–PCR was used to detect any USH1C transcripts (green arrows) and for discriminating USH1C_a and _c splice variants (blue arrows) from USH1C_b3 (orange arrows).
  2. USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs, except in the heart and cerebellum. RT–PCR for GAPDH was used as control (Ctrl.).
  3. USH1C_a or _c splice variants were detected in the retina, kidney and intestine (blue arrows). Electropherogram from the duodenum, confirm alternative splicing of exon 13, but not exon 11. USH1C_b variants were also detected in all examined organs, with presumably two splice variants (orange arrows). Electropherogram from the colon, indicates alternative splicing of exon 20 as presumable reason for the double band. Exon 27 was not detected in any amplicon of USH1C_b RT–PCR (Appendix Fig S10D and E). Sequencing was done with a forward primer.
  4. Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks (3w) (1 piglet, 1 retina, 1 duodenum) and 1 year (1y) (2 pigs, 1 retina each, 3 TRs). Arrow indicates the expected size of harmonin isoform a (72 kDa).
  5. Representative immunofluorescence staining of harmonin (green) of longitudinal cryosection through WT and USH1C pig retinas (2 pigs, 1 y, 1 retina each, 3 TRs, scale bar 10 µm) demonstrated the absence of harmonin staining in the outer segment (OS) of photoreceptor cells and in the other layers below the outer limiting membrane (OLM, arrow) of the USH1C pig retina.
  6. Representative Western blot analysis of retinal protein extracts, indicating that the abundance of the USH proteins SANS (USH1G), whirlin (USH2D) or myosin7a (USH1B) is unaffected (1 piglet, 3w, 1 retina each, 2 TRs).