Pbudce4

Cloning of luciferase reporters for miRNA binding sites was done as previously described^{16 (link)}. Briefly, to express different pre-miRNAs, a mammalian expression vector pBudCE4.1 was used (Thermo Fisher). PCR primers were designed to amply the genomic regions flanking pre-miRNAs (~200-nt upstream and downstream). Mouse mir124-1 was cloned downstream of the CMV promoter between Hind III and BamH I to serve as a control for transfection and expression experiments. Pre-miR-217-WT was cloned downstream of the EF-1α promoter between Not I and Kpn I. All other variants of pre-miR-217 were made using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent). All oligo sequences for cloning can be found in Supplementary Data 6. The dual-luciferase reporter assay was performed according to our published protocol^{16 (link)}, with the exception that luciferase activities were measured 48 h after cotransfection of 100 ng pre-miRNA plasmids and 100 ng corresponding reporter plasmids.

The DNA coding sequence without the stop codon and HindIII and XbaI linkers of the wild-type and mutated human GNAT1 were synthesized in an optimized way and cloned in a mammalian expression vector pBudCE4.1 (Thermo Fisher, Villebon-sur-Yvette; GeneCust, Dudelange, Luxembourg). This vector contains a C-terminal myc tag, which allows detecting the protein by immunolocalization with an anti-myc antibody in case the antibody directed against the endogenous protein is not working. Transient transfection studies were performed in COS-1 cells similarly as previously described [44 (link)]. To detect the protein cells were stained with either mouse anti-GNAT1 antibody (sc136143, Santa Cruz Biotechnology, CliniSciences, Nanterre, France) or anti-myc antibody (11667149001, Roche, Basel, Switzerland) and secondary anti-mouse Cy3 mouse antibody (711-165-150, Jackson ImmunoResearch Laboratories, Baltimore, MD, USA). Subsequently cells were stained with DAPI (40,6-diamidino-2-phenylindole) (AAT Bioquest, Sunnyvale, Etats Unis) and mounted in mounting medium (Fluoromount-G, Southern Biotech, Birmingham, AL, USA) using coverslips. Cell preparations were visualized with standard fluorescence microscopy (DM6000, Leica, Wetzlar, Germany) at a 60x magnification.

The cDNA of GRIN1 was kindly provided by Prof. Dr. Wanker (MDC, Berlin) and cloned into pBudCE4.1 (Life Technologies). NR1 DNA (1 μg) was mixed with 3 μg PEI and 100 μl 150 mM NaCl, vortexed and incubated for 10 min, and HEK293 cells were transiently transfected. Two days later, HEK cells on cover slips were fixed with methanol at −20 °C for 4 min. In addition, HEK cells transfected with a different NMDAR clone, leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein 2 (Caspr2), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, and gamma-aminobutyric acid b (GABAb) receptor were used (Autoimmune-Enzephalitis-Mosaik 1, Euroimmun, Lübeck, Germany). For staining with polar bear CSF, cells were washed in PBS, preincubated with 5% normal goat serum containing 2% bovine serum albumin and 0.1% Triton X-100, and incubated with CSF starting at a 1:5 dilution overnight at 4 °C. Sections were washed in PBS and coverslips mounted with Immu-Mount (ThermoScientific). No polar bear serum was available for antibody detection as blood from Knut was coagulated and autolysed as described1 (link). Double-labeling of transfected cells was performed using commercial antibodies: monoclonal mouse anti-NR1 (1:100, Synaptic Systems).

Human EPO sequence was retrieved from Uniprot P01588 (EPO_HUMAN), PCR amplified, and cloned into pcDNA3.1(+) vector using NheI and EcoRI sites. A codon-optimized gene for EPO (coEPO) was ordered from GeneArt and cloned in the same manner. The synthetic sequences of Rituximab heavy and light chain were ordered codon optimized for CHO from GeneArt. Sequences were based on protein sequences from the DrugBank database under accession number DB00073. An N-terminal signal peptide (MGWSCIILFLVATATGVHS) was added to both protein sequences. Rituximab heavy chain and light chain were cloned into the dual expression vector pBudCE4.1 (Life Technologies, Carlsbad, CA, USA). Empty vector control was pcDNA3.1(+). Rituximab heavy chain was PCR amplified and cloned using SalI and BamHI, and Rituximab light chain was PCR amplified and cloned using NotI and XhoI.

The CDS of GCaMP6s was amplified from HBT-GCaMP6-HA^{48 (link)} and cloned into a dual-promoter vector, pBudCE4.1 (Invitrogen), with each CDS for MLOs or ANX1/BUPS1/LLG2/MARIS for co-expression in HEK293T or COS7 cell.
Mammalian cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO₂ incubator at 37 °C with controlled moisture. HEK293T or COS7 cells were transfected using Lipofectamine™ 3000 Transfection Reagent Kit (Invitrogen). Plasmids for transfection were extracted from E. coli (DH5α) using QIAGEN Plasmid Mini Kit (Qiagen), and 2 μg plasmid DNA was added into each well of 6-well plates (Nunc) containing the cells (70%–80% confluent). To confirm that the cells were successfully transfected, green and/or red fluorescent signals were examined using an inverted fluorescence microscope (Zeiss AxioObserver Z1 Inverted Microscope) before patch clamp and Ca²⁺ imaging experiments were performed 48 h after transfection.

The pcDNA3 plasmid containing WT NPM-ALK was obtained from the original pSRα-tkneo-NPM-ALK plasmid (a kind gift from Dr. S. Morris, S. Jude Research Hospital, Memphis, TN, USA), whereas all the other mutants were generated by site-directed mutagenesis, using the Phusion site-directed mutagenesis kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). To co-express WT and mutant NPM-ALK kinase in COS7 or HEK-293T cells, a double cassette vector pBudCE4.1 (Invitrogen) was used, in which WT NPM-ALK was subcloned into the EF-1α multiple cloning site (MCS) and fused to the V5 epitope, whereas NPM-ALK mutants were subcloned into the CMV MCS and fused to myc tag.
The full-length human ALK cDNA was purchased from ATCC and subcloned into the mammalian expression vector pcDNA3.1. Point mutations F1174L and R1275Q were introduced by site-directed mutagenesis, as previously indicated.