FIG 5.

Deletion of the p66^Shc promoter results in a monoallelic-to-biallelic transcriptional switch of p52^Shc. (A, top) Positions of sgRNAs used in CRISPR/Cas9 and PCR primers used for the PCR assay; (bottom) PCR products generated with the indicated primers. WT, normal HUVECs; Δp66P/Δp66P, HUVECs harboring homozygous p66^Shc promoter deletions. (B) Immunoblots of Shc1 and actin in normal HUVECs and p66^Shc promoter-deleted HUVECs. (C and D) 3C was performed to assess the change of chromatin configuration of SHC1 when the p66^Shc promoter was deleted, with the DNA fragment harboring the p52^Shc promoter (C) or the p66^Shc promoter (D) as anchor. Error bars indicate means ± SD for three 3C experiments. (E) Representative traces of Sanger sequencing of RT-PCR products of p52^Shc containing the informative exonic SNP (arrow) in normal HUVECs and p66^Shc promoter-deleted HUVECs. (F) Quantitative analysis of Sanger sequencing of RT-PCR products of p52^Shc containing the informative exonic SNP revealing alleles transcribing p52^Shc in single HUVECs with the p66^Shc promoter deletion. (G) Transcription of p52^Shc in HUVECs and HUVECs with p66^Shc promoter deletion, evaluated by quantitative and nanofluidic digital PCR assay.