Pcdna3 ha ubiquitin

Berberine chloride, protein synthesis inhibitor cycloheximide and proteasome inhibitor MG-132 were purchased from Sigma-aldrich (St. Louis, MO, USA). Plasmid pcDNA3 Cyclin D1-HA (Plasmid 11181) and pcDNA3 Cyclin D1-HA (T286A, Plasmid 11182) were kindly provided by Bruce Zetter (Harvard Medical School, deposited by Addgene, Cambridge, MA, USA); plasmid pcDNA3 HA-ubiquitin (Plasmid 18712) was provided by Edward Yeh (The University of Texas-Houston Health Science Center, deposited by Addgene).

pcDNA 3.1 (-) Myc-His-human PKM2, -mouse PKM2, pcDNA 3.1 Flag-human PKM2, -HSP90, pcDNA 3 HA-ubiquitin, and pBABE-bleo IGF-1R (Addgene, Watertown, MA, USA #11212) were used for overexpression experiments. The AccuTarget™ Negative Control siRNA (siCon, Bioneer, Daejeon, Korea), On-TARGET plus non-targeting pool siRNA, and custom-designed siPKM2 (Dharmacon, Lafayette, CO, USA, #D0018101050) were used in the gene-silencing study. The negative and non-targeting pool siRNAs had no significant homology to any known human gene sequences. For the transfection experiments, cDNA vectors and siRNAs were transfected using Lipofectamine^® 2000 reagent (Invtrogen, Carlsbad, CA, USA, #11668019) or Lipofectamine^® RNA-iMAX (Invitrogen, #13778075), respectively, according to the manufacturer’s instructions. Briefly, Lipofectamine^® reagents and cDNA or siRNA (20 nM) were mixed and incubated in Opti-MEM (GIBCO, #31-985-070) at room temperature for 15 min to form transfection complexes. Subsequently, the mixtures were added to each well, and then the cells were further incubated for 48h.

Mouse Tmem79 was tagged with Myc or Flag or both at the C-terminus. FZD1-10 were tagged with 2xHA or V5 epitope right after the signal peptide. HA-USP8 was generated by PCR-subcloning using Flag-HA-USP8 (Addgene, #22608) as template. V5-FZD5-K0 was generated by site-directed mutagenesis (cytoplasmic lysines 347, 434, 439, 442, 445, 525, 546 and 578 were substituted with arginines). ZNRF3-HA was a gift from Dr. Feng Cong. HA tag in pcDNA3-HA-Ubiquitin (Addgene, #18712) was replaced with 6xHis by PCR cloning to make His-Ubiquitin. Xenopus laevis tmem79 and usp8 cDNAs were PCR amplified from Xenopus stage 10 cDNA library and cloned into pCS2+ vector. SuperTopflash vector 7TFP was obtained from Addgene (#24308). Renilla was PCR amplified from pRL-TK (Promega) and subcloned into lentiviral vector pHage-EF1α. To make lentiviral Top-EGFP reporter plasmid, 7xTCF promoter and EGFP were amplified from 7TFP and pEGFP-N1 (Clontech) vectors, respectively, and subcloned into lentiviral vector pHage.