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. 2020 Nov 3;21(21):8222. doi: 10.3390/ijms21218222

Figure 4.

Figure 4

(A) RT-PCR demonstrating MCPyV VP1, small t-antigen (tag) and large T-antigen (Tag) (left panel), and 18s rRNA, exon 6 and 7 containing (exons 6/7 TrkA) and noncontaining (exons 5/8, TrkAIII) products generated from metastatic MCPyV-positive MCC RNA. (B) RT-PCR demonstrating (left panel) a 2372-bp product expected for full-length fully spliced TrkA and a 2096-bp product expected for Δ exon 6–7 TrkAIII generated from metastatic MCC RNA using primers spanning TrkA exons 1 to 17, and (right panel) a 1112-bp product expected for fully spliced TrkA exons 1–8, an 836-bp product expected from Δ exon 6–7 TrkAIII exons 1–8, a yet to be characterised product (?) and the 500-bp product characterised as Δ exon 2-7 TrkAIV, generated from metastatic MCPyV-positive MCC RNA, using primers spanning TrkA exons 1 to 8, plus a unique 1280-bp product expected for fully spliced TrkA exons 10–17, generated from metastatic MCPyV-positive MCC RNA using primers spanning TrkA exons 10 to 17. (C) Gel-purified 1112-bp (fully spliced TrkA), 836-bp (Δ exon 6–7 TrkAIII) and 500-bp (Δ exon 2-7 TrkAIV) TrkA exon 1 to 8 RT-PCR products (left panel), and (right panel) RT-PCR confirmation of Δ exon 6–7 TrkAIII and TrkA identity of TrkA and D exon 6–7 TrkAIII control cDNAs, gel-purified 1112-bp TrkA exon 1–8 and 836-bp Δ exon 6–7 TrkAIII RT-PCR products, using TrkA and Δ exon 6–7 TrkAIII-specific primers. (D) Sequence confirmation of the novel Δ exon 6–7 TrkAIII exon 5/8 splice junction in the gel-purified 836-bp Δ exon 6–7 TrkAIII exon 1–8 RT-PCR product (3rd panel), and confirmation of the novel exon 1/8 splice junction (green) in the 500-bp Δ exon 2–7 TrkAIV exon 1–8 RT-PCR product (4th panel), plus a section of nucleotide and potential amino acid sequence for Δ exon 2–7 TrkAIV demonstrating frame-shift induced tga stop codons (red). (E) Western blot demonstrating 140 kDa and 100 kDa (arrows) TrkA immunoreactive species in protein extracts from the MCPyV-positive metastatic MCC (MCC) but not in a normal skin extract (100 μg loads), compared to 140 kDa fully spliced TrkA and 100 kDa Δ exon 6–7 TrkAIII in stable TrkA and Δ exon 6–7 TrkAIII transfected SH-SY5Y cell extracts but not in stable empty pcDNA vector-transfected SH-SY5Y cell extract negative control (Cont; 20 μg loads).