4.2. Plasmids and shRNA

shUSP1 constructs were generated in the pLKO.1 vector at the AgeI/EcoRI sites. USP1 overexpression construct was a gift from Wade Harper (Addgene plasmid #22596) and cloned into the pLX304 vector. HA-Ub vector was a kind gift from the Attanasov lab (Roswell Park Comprehensive Cancer Center) and His-Ub was a gift from Dr. Wang’s lab (Roswell Park Comprehensive Cancer Center). TAZ lysine mutants were generated from the pBABE-WT-TAZ vector using a site-directed mutagenesis kit (New England Biolabs, Boston, MA, USA) and PCR. Sequences for site-directed mutagenesis are found in Table 1. shRNA hairpins targeting TAZ were obtained from Broad Institute RNAi consortium and target sequences are in Table 1. Flow cytometry was used to isolate GFP positive TAZ overexpressing cells.

Key resources table.

Lentiviral packaging: Briefly, shRNA plasmid, Δ8.9 and VSVG were co-transfected into 293T cells with Fugen transfection reagent. Viral supernatants were collected on day 3 and 4 after transfection.