Pepmute reagent

The Nrdp1-FLAG and N-Nrdp1–FLAG constructs have been previously described (12 (link)), as have the C34S/H36Q mutation of Nrdp1 (11 (link), 31 (link)), the coiled-coil deletion mutant (35 (link)), and the shNrdp1 construct KD1 (13 (link), 38 (link)). Rtn1A complementary DNA (cDNA) was purchased from ATCC and sub-cloned into pcDNA3.1(+). GFP-Sec61β, GFP-DP1, and GFP-Rtn4AHD were gifts of the T. Rapoport laboratory (Harvard Medical School) (26 (link)). Re-ticulon domain deletion mutants were subcloned into pAcGFP1-C1 and contain Rtn4A amino acids 916 to 1018 (N-cyto), 1017 to 1054 (TM1), 1053 to 1121 (Loop), 1120 to 1151 (TM2), or 1150 to 1192 (C-cyto). Cells were transfected with the PolyJet reagent (SignaGen Laboratories) following the manufacturer’s instructions with equal amounts of each plasmid and pcDNA3.1 or pSuper/Scramble as the vector control. siRNA directed toward human Rtn4A contained the recognition sequences 5’-ACCCAAAGUU-GAAGAGAAA-3’ (KD1) and 5’-GGUAAUUUGUCAACAGUAU-3’ (KD2) and was transfected into cells using PepMute reagent (SignaGen Laboratories). Cells were treated with 15 µM siRNA for 5 days (MCF10AT and C2C12) or 6 days (MCF7). siRNA directed toward mouse Rtn4A contained the sequence 5’-CGAAAGAAGCAGAGGAAAA-3’ and was transfected into cells using DharmaFECT 1 reagent (Dharmacon).

The cells were transfected with siRNA using PepMute reagent (SignaGen Laboratories, USA) after reaching 80–90 % confluence. The Genjet Plus reagent (SignaGen Laboratories, USA) was used for plasmid transfections for gene over-expression. The V5 tagged USP13 plasmid was generated in the laboratory previously. The Flag tagged USP13 plasmid was a gift from Dr. Ze’ev Ronai, Director of Sanford Burnham Prebys Medical Discovery Institute (La Jolla, CA, 92037 USA). Human MDM2 Gene ORF cDNA clone expression plasmid was purchased from Sino Biological US (Cat. No.: HG11206-NY). All siRNAs used in the experiments were purchased from Sigma.