Pegfp n1

The vector plasmid gWiz Blank was commercially prepared (Aldevron, Fargo, ND, USA) at concentration of 2 mg/ml in physiological saline. Additionally, concentrations of 1 mg/ml by further dilution and 3.5 mg/ml by concentration (Concentrator plus, Eppendorf, Hamburg, Germany) were prepared.
Plasmid EGFP-N1 (pEGFP-N1, BD Biosciences, San Jose, CA, USA), encoding green fluorescent protein, was used for transfection efficiency experiments. It was isolated after amplification in a competent Escherichia coli (TOP10; Thermo Fisher Scientific) using Maxi-Endo Free Plasmid Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of isolated plasmid was measured with Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA).

pCS2HA-βTrCP, pCS2HA-βTrCPΔF and empty vectors have been previously described [29 (link)-31 (link)]. pEGFP-N1 and pRBG4-MT-Ub were from BD Biosciences and ATCC, respectively. pFlagCMV2-CDK1 and pCMVHA-FBXW7α were obtained by cloning the corresponding PCR fragments in pFlagCMV2 and pCMVHA, respectively. Flag-CDK1 βM was constructed using the “Transformer Site-Directed Mutagenesis Kit” from BD Biosciences. Construct sequences and point mutations were verified on both strands with an automatic sequencer.

The green fluorescence protein (GFP)-tagged human ErbB-2 mutant, which lacks the putative nuclear localization signal (NLS) (aa 676-KRRQQKIRKYTMRR-689; hErbB-2ΔNLS) [13 ], was generously provided by Dr. MC Hung (University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA). The expression plasmid encoding GalNAc-T-Cherry, a Golgi complex marker [58 (link)], was kindly gifted by Dr. Daniotti (CIQUIBIC-CONICET). The empty vector pEGFP-N1 was obtained from BD Biosciences-Clontech. Site-directed mutagenesis was performed on the WTErbB-2-pEYFP to produce the deletion of a region containing leucines 11–13 (ErbB-2∆11–13-pEYFP, ErbB-2∆11–13), using the following primers: 5′-GGCAAGAGGGCCCCCCAGCGGC-3′ and 5′-GCCGCTGGGGGGCCCTCTTGCC-3′. Successful mutation of the sequence was verified by DNA sequencing at Macrogen (Seoul, SK). Cells were transfected for 72 h with 2 µg of expression vectors using X-tremeGENE HP (Roche) following the manufacturer’s instructions. In the case of the GalNAc-T-Cherry plasmid, cells were transfected for 48 h before treatment with R2. Transfection efficiencies, evaluated using the pEGFP-N1 vector and determined by the percentage of cells exhibiting GFP 96 h after transfection, varied between 60 and 70%.

A wild type 3′-UTR of TMEM182 containing the miR-450a binding sites (3'UTR-WT) and truncated 3′-UTR fragment with deleted miR-450a binding sites (3'UTR-DEL) were constructed into the XhoI/XbaI sites of pmiRGLO firefly luciferase-expressing vector (Promega, WI, USA). For gene knockdown experiments, the shRNA clones of TMEM182 (sh182 #1 & #2) and empty vector pLKO_TRC (shCTRL) were obtained from the National RNAi Core Facility (Academia Sinica, Taiwan). Human TMEM182 cDNA was sub-cloned into empty vector pCDH-CMV-GFP puro+ (vehicle) (System Biosciences) at EcoRI/BamHI sites and termed as TMEM182-flag. Human TMEM182 cDNA was sub-cloned into empty vector pEGFPN1 (BD Biosciences Clontech’s) (vehicle) at XhoI/BamHI sites, termed as TMEM182-GFP. The sequence data were compared against the National Center for Biotechnology Information (NCBI) database using BLAST and miRBase (http://www.mirbase.org/). Kyte-Doolittle hydrophobicity analysis was accessed to predict TMEM182 transmembrane portions using ExPASY (https://web.expasy.org/protscale/). List primer sequences in the following S2 Table.

The primers for zebrafish IRF1 and RelA were designed according to sequences in Ensembl (http://www.ensembl.org). Total RNAs were extracted from zebrafish spleen by using TRIzol Reagent (Invitrogen) and reverse-transcribed into cDNAs, then IRF1 and RelA cDNAs were generated through RT-PCR. The PCR products were purified from 1.2% agarose gel by using gel extraction kit (Omega), and inserted into the pcDNA6 (Invitrogen) and pEGFP-N1 (BD Biosciences) vectors. The plasmids were transformed into competent Escherichia coli DH5α (Invitrogen), and the positive plasmids were purified by using endo-free plasmid minikit II (Omega Bio-tek).

The primers for zebrafish IRF1 and RelA were designed according to sequences in Ensembl (http://www.ensembl.org). Total RNAs were extracted from zebrafish spleen by using TRIzol Reagent (Invitrogen) and reverse-transcribed into cDNAs, then IRF1 and RelA cDNAs were generated through RT-PCR. The PCR products were purified from 1.2% agarose gel by using gel extraction kit (Omega), and inserted into the pcDNA6 (Invitrogen) and pEGFP-N1 (BD Biosciences) vectors. The plasmids were transformed into competent Escherichia coli DH5α (Invitrogen), and the positive plasmids were purified by using endo-free plasmid minikit II (Omega Bio-tek).

The plasmid pEGFP-N1 was purchased from BD Biosciences (San Jose, CA). It was transformed into competent DH5α cells, and plated onto LB plates supplemented with 100 μg/mL ampicillin. Afterwards, it was purified with the Plasmid Giga Kit (Quiagen, Valencia, CA) according to the manufacturer's guidelines. The quality and quantity of the purified plasmid were analyzed by measuring its optical densities at 260 and 280 nm.