Shrna lentiviral plasmids plko 1 puro

β-TrCP2-small interfering RNA (siRNA) (5′-GAGCUCUUGGUGGAUCAUCTT-3′), control siRNA duplex (Dharmacon, CO), and GSK-3α and GSK-3β siRNA expression plasmids (Upstate Biotechnology) were transfected with Lipofectamine 2000 (Invitrogen, CA) or electroporation using Nucleofector 1 (Amaxa Biosystems, MD). The shRNA lentiviral plasmids (pLKO.1-puro) against human β-TrCP were purchased from Sigma. The sequences targeting β-TrCP1 are 5′-CCGGGCACATAAACTCGTATCTTAACTCGAGTTAAGATACGAGTTTATGTGCTTTTT-3′ and 5′-CCGGGCGTTGTATTCGATTTGATAACTCGAGTTATCAAATCGAATACAACGCTTTTT-3′; the sequences targeting β-TrCP2 are 5′-CCGGAGAAGACTTGGCCTCTAATTTCTCGAGAAATTAGAGGCCAAGTCTTCTTTTTTG-3′ and 5′-CCGGTATCAGTGGCCTACGAGATAACTCGAGTTATCTCGTAGGCCACTGATATTTTTG-3′. HEK 293T cells were transfected with indicated lentiviral vector. 24 hours later, puromycin (1g/ml) was added to the cells to select puromycin-resistant clones.

Reduction in frataxin in cultured DRG neurons was achieved by short‐hairpin RNA‐interfering sequences. Lentiviral vectors are routinely produced as described in our previous publication 24. The shRNA lentiviral plasmids (pLKO.1‐puro) for human/mouse/rat frataxin were purchased from Sigma‐Aldrich (Merck. Madrid. Spain). The RefSeq used was NM_008044, which corresponds to mouse frataxin. The clones used were TRCN0000197534, TRCN0000006137 and referred in this paper as Fxn1 and Fxn2, respectively). A non‐targeted scrambled sequence (the vector SHC002) served as a control (here referred as Scr).