Peyfp n1

Madin-Darby Canine Kidney (MDCK) cells were stably transfected with a plasmid encoding human full-length keratin eight with an EYFP tag at its carboxyterminus (Windoffer et al., 2004 (link); recloned into pEYFP-N1 (Clontech) with BamHI and EcoRI). MDCK cells were obtaned from DKFZ (Deutsches Krebsforschungszentrum). Resulting cell clone H9 was used for the analyses described in this paper. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) with 10% (v/v) fetal calf serum (Pan Biotech) including 700 µg/ml G418 (Sigma-Aldrich) for selection. Vital MDCK cells were imaged after 3 days when the monolayer had reached complete confluence.
Immortalized human HaCaT keratinocyte cell clone B10 expressing EYFP-tagged human keratin 5 (HK5-EYFP) has been described (Moch et al., 2013 (link)). HaCaT cells were obtained from the Fusenig lab, which first isolated and described this cell line (Boukamp et al., 1988 (link)). The cells were grown in DMEM containing l-alanyl-glutamine (Sigma-Aldrich) supplemented with 10% (v/v) fetal calf serum (SeraPlus; PAN Biotech) and passaged as described (Moch et al., 2013 (link)). Vital HaCaT cells were imaged 1–2 days after reaching confluence corresponding to 5–7 after seeding. Both cell lines were tested by PCR to be mycoplasma free.

The human cDNAs for the D₃, MT₁, MT₂, and GHS-R1a receptors cloned in pcDNA3.1 were amplified without their stop codons using sense and antisense primers. The primers harbored either unique BamHI and HindIII sites for D₃; EcoRI and KpnI sites for GHS-R1a or Hind III; and BamH1 sites for MT₁ and MT₂. The fragments were subcloned to be in frame with an enhanced yellow fluorescent protein (pEYFP-N1; Clontech, Heidelberg, Germany) or an Rluc (pRluc-N1; PerkinElmer, Wellesley, MA) on the C-terminal end of the receptor to produce D₃-RLuc, MT₁–YFP, MT₂–YFP and GHS-R1a-YFP fusion proteins.

Human Kv2.1 (GenBank Accession number NM_004975) and human Kv6.4 (NM_172347) in the eGFP-N1 vector (Clontech, Palo Alto, CA, USA) and N-terminally CFP-labeled Kv2.1 and Kv6.4 subunits in the eCFP-C1 vector (Clontech) have been described previously14 (link)17 (link). Mouse KCNE4 (NM_021342) was amplified from mouse genomic DNA and subcloned in the pBK vector using SmaI. Human KCNE5 (NM_012282) was amplified from human genomic DNA and subcloned into either the pBK vector using SalI and BamHI or into the pXOOM vector using BamHI and EcoRI. C-terminally YFP-labeled KCNE5 was obtained by subcloning the KCNE5 cDNA in the peYFP-N1 (Clontech) vector. HA-Kv6.4 and HA-KCNE5 constructs were generated by introducing the HA epitope (YPYDVPDYA) into the extracellular S1-S2 loop and the extracellular N-terminus, respectively, by PCR amplification using the QuikChange^TM Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) and mutant primers. All PCR-generated inserts were sequenced to confirm sequence integrity and in-frame clonings.

Human AQP5 and rat AQP5 were obtained from Dr. Bruce Baum (NIDCR, NIH) and subcloned in the following vectors pEGFP-C1, pEGFP-N1, pEYFP-C1, pEYFP-N1 (Clontech). TGN38-mCherry and YFP-TGN38 were a generous gift from Dr. Sarah Hamm-Alvarez (UCSC). GFP-actin, GFP-myosin Vc and GFP-TfnR were obtained from Dr. Julie Donaldson (NHLBI, NIH). GFP-Ribonuclease was a generous gift from Dr. David Yule (Rochester University). The 2X-EYFP vector was generated by inserting a copy of the YFP into a pEYFP-C and used to generate the 3X-EYFP using the same procedure. HSG-cell lines stably expressing YFP-AQP5 were prepared by lentiviral expression, as previously reported (Amornphimoltham et al., 2013 (link)).

All reagents used were of molecular biology grade, purchased from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise. HEK293 cells stably expressing human RyR subtype 2 (HEK293_RyR2) and D1ER constructs were provided by Roland Malli (Medical University of Graz, Graz, Austria). We cloned TRIC-A fusion constructs in pCMV-Myc, pECFP-N1, and pEYFP-N1 vectors (Clontech, Saint-Germain-en-Laye, France) from untagged mouse TRIC-A, provided by Hiroshi Takeshima (Kyoto University, Kyoto, Japan). YFP-STIM1, Orai1-CFP, STIM1-myc, mCerulean-ER-5, and mCherry-ER-3 were obtained from Indu Suresh Ambudkar (NIH, Bethesda, MD, USA). STIM1-CFP, STIM1-YFP, CFP-STIM1, YFP-Orai1, YFP-Orai1-E106Q, and YFP-STIM1-D76A were provided by Christoph Romanin (University of Linz, Linz, Austria). CFP-GPI was provided by Wolfgang Schreibmayer (Medical University of Graz, Graz, Austria).