P3xflag cmv 10 expression vector

Expression plasmids for untagged and HA-tagged Def6 were generated as described previously^{21 (link)}. Expression constructs for Myc-tagged raptor (Addgene plasmid 1859^{53 (link)}) and HA-tagged p62 (Addgene plasmid 28027^{54 (link)}) were purchased from Addgene. Expression plasmids for Flag-tagged TRAF6 and Flag-tagged full-length p62 and its various deletion mutants were constructed in p3XFLAG-CMV-10 expression vector (Sigma) and transfected into 293T (CRL-3216; ATCC) cells.

Myc-tagged BRCA1 expression vectors used for co-IP assay were constructed with pcDNA6/V5-His expression vector (Thermo Fisher Scientific, Waltham, MA, USA) without any tags at the C-terminal. FLAG-Parkin expression vectors used for co-IP assay were constructed with p3xFLAG-CMV-10 expression vector (SIGMA). Knockout vectors for PINK1 were constructed with pSpCas9(BB)-2A-Puro (PX459) V2.0 vector (gift from Dr. Feng Zhang: Addgene # 62988)^{45 (link)} using the sequence shown in Table S2.

The entire open reading frame of the murine SRP68 gene (NM_146032.3) was amplified by PCR using cDNA derived from C2C12 cells as a template. The resulting PCR product was digested with EcoRI and BamH1 and cloned into the p3xFlag-CMV10 expression vector (Sigma). The primer sequences are shown in Table B in S1 File. The resulting construct or the empty vector were transfected into C2C12 cells as described above [31 (link)] and increased expression of SRP68 was verified by western blot 24 hrs later.

18E6WT Open Reading Frame (ORF) was obtained and PCR-amplified from an HPV-18 positive cervical cancer biopsy, 18E6*I ORF was obtained by RT-PCR amplification from HeLa cells (HPV-18 positive), and 16E6 ORF was amplified by PCR from CaSki cells (HPV-16 positive). The HPV-18 E6 spliced mutant (18E6SM) sequence was amplified from the plasmid, pCAHPV18-E6sm [32 (link)]. 18E6SM harbors a mutation at the donor splicing site (G233A), favoring the expression of the E6 full-length. The HPV-18 E6Af variant (from African phylogenetic branch) and the E6AsAi variant (from Asian-Amerindian phylogenetic branch), which is the canonical reference variant, were PCR-amplified from DNA previously obtained from tumor biopsies [27 (link)]. All these fragments were purified and cloned into the p3x-FLAG CMV.10 expression vector (Sigma Aldrich, Sant Louis, MO, USA) Constructs were verified by DNA-sequencing. β-catenin, pCAHPV18-E6sm, and pGW1-18E6-HA (hemagglutinin-tagged) expressing plasmids were kindly provided by Lawrence Banks (ICGEB, Trieste, Italy). The TCF-4 reporter plasmid (TOPFLASH) containing two sets of 3 copies of TCF-4 binding sites upstream of Thymidine Kinase minimal promoter and luciferase ORF (Merck-Millipore, Burlington, MA, USA) was used to perform luciferase assays, and pCMV-β-galactosidase plasmid (Promega, Madison, WI, USA) was used to evaluate the efficiency of transfection.

The Flag-tagged full-length FLAG-NR4A1 expression plasmids were constructed by inserting PCR-amplified full-length NR4A1 (amino acids 1–599) into the EcoRI/BamHI site and C-terminal NR4A1 (amino acids 67–599) and N-terminal NR4A1 (amino acids 1–354) into the EcoRI/KpmI site of the p3XFLAG-CMV-10 expression vector (Sigma-Aldrich). NBRE_x3-luc was generously provided by Dr Jacques Drouin (University of Montreal, Montreal, QC, Canada). All other plasmids used in this study were previously described [21 (link),22 (link)].

ROS cells were cultured in DMEM/F12 (1:1), L-glutamine, 15 mM HEPES (Gibco Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (Gibco Thermo Fisher Scientific), penicillin and streptomycin. MEFs were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin and streptomycin (Gibco Thermo Fisher Scientific). Cells were passaged when confluent with 0.05% trypsin/EDTA and maintained at 37 °C and 5% CO₂. ROS cells and MEFs were transfected with wild type Ank, mutant Ank with deletion of TTC_1130–1132 (phenylalanine at position 377, F377del) or deletion of TCC_1124–1126 (serine at position 375, S375del) in p3xFLAG-CMV-10 expression vectors (Sigma-Aldrich) using JetPEI (Polyplus transfection). Stable ROS cell clones expressing the gene construct for wild type and mutant Ank were selected by incubating in G418 selection medium. Expression of FLAG-ANK in stably transfected ROS cells was confirmed by PCR followed by immunoblots with FLAG and ANK antibodies. Vectors pRK5-HA-Ubiquitin-KO, pRK5-HA-Ubiquitin K48R, SNAP-3xFLAG-Ank, CLIP-3xFLAG-Ank were transfected using the same method.