Pcdna3.1 flag vector

Murine GnT-II (Mgat2) cDNA from osteoblasts were inserted into pcDNA3.1(+)/FLAG vector (Invitrogen) at BamHI and EcoRI sites. The cDNA was synthesized with a primer set: BamH1, 5′-ATA​GGA​TCC​ATG​AGG​TTC​CGC​ATC​TAC​AAA​CG-3′; EcoRI, 5′-ATA​GAA​TTC​CTG​CAG​TCT​TCT​ATA​ACT​TTT​ACA​GAG​TTC​ATG​G-3′. We transfected the vector into HEK293T cells by Lipofectoamine LTX (Invitrogen) and incubated in Opti-MEM for 24 h. The transient Mgat2-293T cells were harvested with trypsin digestion and resuspended in a TSA buffer for the FACS analysis.

Complementary DNAs encoding human SRPK1 were amplified by polymerase chain reaction using a cDNA library prepared from the human fetal brain as the template and then inserted into the pcDNA3.1 FLAG vector (Invitrogen). The vector expressing a SRPK1-targeting small hairpin RNA was constructed as follows. The short hairpin primers were designed around the potential sequence, namely sh1-SRPK1 (5′-GTGCAGCAGAAATTAATT-3′), which corresponds to nucleotides 1171–1189 of the SRPK1 transcript (GI: 47419935), and the sequence was annealed and then ligated into the PstI site of the pSilencer1.0 U6 vector (Ambion). The MCL-1 minigene reporter was constructed by inserting a complete human MCL-1 genomic fragment containing exons 1, 2, and 3 and intra-exon introns. The genomic fragments were amplified by PCR using the genomic DNA prepared from MRC5 fibroblasts as the template and then inserted into the HindIII/SacI sites to replace the β-galactosidase gene of pCH110 (Amersham Pharmacia). Mutant pCH-MCL-1 vectors containing changed nucleotides were constructed using the QuikChange site-directed mutagenesis system (Stratagene).

The N-terminal (NT) domain, the FH1 and FH2 regions and the mutant of FMNL2 were amplified respectively by PCR utilising the primers (Supplementary Table S1) and inserted into pCDNA3.1-FLAG vector (Invitrogen, Foster city, CA, USA). The complete sequence in open reading frame of COMMD10 was amplified by PCR using the primers (Supplementary Table S1) and subcloned into the pEGFP-C1 vector at the KpnI-XhoI site. The C-terminal (CT) and NT domains were amplified by PCR using the primers (Supplementary Table S1) and the fragments of COMMD10 digested with BamHI and XhoI were subcloned into the pEGX-6p-1 vector at the BamHI-XhoI site. The PCR conditions were as follows: 95 °C for 3 min, followed by 30 cycles of amplification (94 °C for 30 s, 55 °C for 40 s with outer primers or 68 °C for 40 s with inner primers, and 72 °C for 2 min). The correct coding regions of all plasmids were confirmed by sequencing. Transient transfection was performed using Lipofectamine2000 as a vehicle according to the manufacturer’s instructions (Invitrogen, Beijing, China). Lentiviral construct expressing or repressing COMMD10 was purchased from GeneCopoeia (Guangzhou, China) and was used to infect CRC cell lines SW480 and HT29. The eukaryotic expression vectors of pENTER-FLAG-RELA and pENTER-FLAG-RELB were purchased from Vigene Biosciences (Shandong, China).