Ha ubiquitin plasmid 18712

bAP15 (11324) was purchased from Cayman Chemical (Ann Arbor, MI). The USP14 inhibitor IU1 (662210) was obtained from EMD Millipore Corporation (Billerica, MA). UCHL5 shRNA plasmid (h) (sc-76797-SH) was purchased from Santa Cruz Biotechnology (Dallas, TX). HA-ubiquitin (Plasmid 18712) was purchased from Addgene (Cambridge, MA).
Antibodies were obtained as follows; anti-β-actin (sc-47778), anti-UCHL5 (sc-271002), anti-USP14 (sc-100630), anti-β-catenin (sc-7963), anti-Vimentin (sc-6260), anti-Ub (sc-166553), and anti-HA-probe (F-7) (sc-7392) (Santa Cruz Biotechnology), anti-E-cadherin (610181), anti-N-cadherin (610920)(BD Bioscience, San Jose, CA), anti-Smad2 (5339), anti-Phospho-Smad2 (3108), anti-Phospho-Smad2 (18338), anti-Smad3 (9523), anti-Phospho-Smad3 (9520), and anti-Smad4 (38454) (Cell Signaling Technology, Danvers, MA).

The full-length construct of Ubiquitin (HA-Ubiquitin Plasmid # 18712) and Nup88 (pEGFP-Nup88 Plasmid # 64283) were obtained from Addgene. The C-terminal coiled-coil domain (Nup88-C, amino acids 585-741) of Nup88 was PCR amplified from human testis cDNA and cloned into pGEX6P1 with EcoRI and SalI enzyme sites. This domain was further subcloned into the pEGFP vector. The Nup88 construct lacking the coiled coil domain (Nup88-delC) was created by inserting a stop codon after 584^th amino acid through site directed mutagenesis (Q5 Site Directed Mutagenesis Kit, NEB-E0554S) using pEGFP-Nup88 as template. pEGFP-Nup62 was a kind gift from Dr Radha Chauhan (NCCS, Pune). The N-terminal and C-terminal truncations of Nup62 were PCR amplified and cloned into pGEX6P1, pCMV3Tag1a and pEGFP vectors. The yeast-two hybrid constructs for Nup88 and Nup62 truncations were made by subcloning them into pGADC1 and pBDUC1 vectors. Nup88-C (aa 585-741) and Nup62-C1 (aa 328-458) coding sequence were inserted into pGADC1 vector harboring GAL4 activation domain (AD) and pBDUC1 vector harboring GAL4 DNA binding domain (BD).