Pires2 egfp

FLAG-tagged TMX1 was generated by PCR using oligos TS470, TS244, TS363, TS364 and TS368 (Fig. S3 A) on a human IMAGE TMX1 EST clone (Invitrogen) by inserting the FLAG-coding sequences to generate a signal peptide cleavage site after glycine 20. The full-length product was cloned into pcDNA3 and pIRES2-EGFP (Takara Bio Inc.) plasmids using the 5′ Kpn1 or the 5′ Nhe1 site, respectively, and the 3′ Xho1 sites. Mutant TMX1 constructs were generated by PCR-based splicing by overlap extension using oligos TS369, TS370 (CCAA) and TS435, TS436 (SXXS). In brief, primary PCR products generated using TMX1 5′ (TS470) and 3′ oligos (TS368) with the mutagenizing upstream and downstream oligos were isolated. Subsequently, a secondary PCR yielded the full-length, mutated cDNA using just the 5′ and 3′ TMX1 oligos. This product was cloned into pIRES-EGFP using the Nhe1–Xho1 sites. The shTMX1 GFP construct (HSH-019885.4-CH1 in psi-H1, target sequence 5′-TCGTGCCAAGCAATAAGAT-3′) was purchased from Genecopoiea. Stealth RNAi HSS129875 (sequence 5′-TGCACAACCTTTGAAAAAAGT-3′) as well as control Stealth RNAi (Medium GC content control siRNA 12935–300) for transient expression were purchased from Invitrogen. The myc-tagged SERCA2b expression plasmid was obtained from U. Petäjä-Repo (University of Oulu, Oulu, Finland).

Experiments were performed using HeLa and N2A cells that were transfected with human Cx40 WT or the mutants A96S, G38D, and M163V cDNA. hCx40 WT or mutant cDNAs were subcloned into the eukaryotic expression vector pIRES2-EGFP (Takara Bio Inc.). Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer’s protocol. The cells were grown in DMEM (Gibco), supplemented with 10% FCS (Hyclone), 100 µg/ml streptomycin (Gibco), and 100 U/ml penicillin (Gibco), and were then passaged weekly, diluted at 1:10, and kept at 37°C in a CO₂ incubator (5% CO₂/95% ambient air). Immunofluorescent staining, electrophysiological measurements, and dye flux studies were performed on cell pairs cultured for 1–3 d.

A cassette containing a One-STrEP-tag (OST) sequence (Junttila et al., 2005 (link)), two STOP codons, an IRES2 sequence (from pIRES2-EGFP; Takara Bio Inc.), a Kozak sequence and a sequence coding for mTFP1, a monomeric, bright, and photostable version of Clavularia cyan fluorescent protein (Ai et al., 2006 (link)), was abutted to a frt-flanked cassette that contains a neomycin-kanamycin resistance (neo^r) gene that can be expressed under the control of a prokaryotic (gb2) or eukaryotic (Pgk1) promoter.

The complete open reading frame of Kcnj13 (the gene encoding Kir7.1) was amplified from a cDNA library, generated by standard procedures from a C57BL/6N male murine small intestine, an organ known to express high levels of Kcnj13. Primers used for the amplification bound to the untranslated region of the gene (in capital letters) with added restriction sites for SacI and SalI (Kir7.1 forward, SacI: 5′-ata​aga​gct​cCA​CAA​AGG​AAC​CGA​GAA​ACA​C-3′; Kir7.1 reverse, SalI: 5′-ata​agt​cga​cGG​ACG​GTG​TAG​ATG​AGT​CTT​A-3′). After restriction digest of the sequence-confirmed full-length Kcnj13 and the mammalian expression vector pIRES2-EGFP (Clonetech; 6029–1) by standard procedures, Kcnj13 was inserted into the vector using the Quick Ligation Kit (New England BioLabs; M2200) according to the manufacturer’s instructions. For recombinant expression, cells of the human embryonic kidney cell line (ATCC; HEK293, CRL-1573) were plated on collagen (Roche; 11179179001)–coated coverslips and transfected using Lipofectamine 2000 reagent (Life Technologies; 11668027) according to the manufacturer’s recommendation. Overexpression was verified by the presence of Kir7.1 in immunocytochemical staining and Western blot analysis as mentioned below. The EGFP of the pIRES2-EGFP vector was used to visualize the transfected cells for whole-cell patch-clamp experiments.