Pis0 vector

A part of the 3′-UTR sequence of TSC1 (935 bp) including the predicted miRNA recognition element (MRE) was cloned into the pIS0 vector (Addgene) containing the luciferase gene (3′-UTR). RT-PCR was used to amplify the 3′-UTR sequence of TSC1 from mRNA isolated from PASMCs using 5′-ATGGAGCTCTGTGTGGAAATGGGACGGAG-3′ and 5′-ATGGGCCGGCCTGGGAAATGATGGTCA-3′. For the 3′-UTR mt, which is the same 3′-UTR with a mutated MRE sequence, an upstream region and a downstream region of the MRE site in the 3′-UTR construct were amplified using primers including the XhoI enzyme recognition sequences. Two PCR products were digested by XhoI enzyme, ligated to change the MRE sequence into the XhoI recognition sequence and then cloned into the pIS0 vector. 5′-ATGGAGCTCTGTGTGGAAATGGGACGGAG-3′ and 5′-TCACTCGAGCAGGTTCAAAGGACGCAAC-3′ were used for the amplification of the upstream region. 5′-TCACTCGAGATGAGGCCAAATTTAATCTTTG-3′ and 5′-ATGGGCCGGCCTGGGAAATGATGGTCA-3′ were used for the amplification of the downstream region. The predicted MRE sequence (23 bp) and MRE mutant (MRE mt) sequence were cloned into the pIS0 vector (Addgene). 5′-GCGTCCTTTGAACCTGTGCAATACCGG-3′ and 5′-TATTGCACAGGTTCAAAGGACGCAGCT-3′ were used for the MRE and 5′-GCGTCCTTTGAACCTGTCGTTAACCGG-3′ and 5′-TTAACGACAGGTTCAAAGGACGCAGCT-3′ were used for the MRE mt.