Pdsred er

cDNAs for rat Eag1 (Dr Olaf Pongs, Saarland University, Germany) and MKRN1 long isoform were subcloned into pcDNA3.1, pcDNA3.1-Myc, or pcDNA3.1-Flag vectors (Invitrogen). Disease-causing (G348R, G469R, and I467V) and glycosylation-deficient Eag1 mutations (N388Q and N406Q; Eag1-QQ), as well as MKRN1 short form and the catalytically inactive E3 ligase mutant MKRN1-H307E, were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies), followed by verification with DNA sequencing. The other cDNA constructs include pDsRed-Monomer-Membrane (Clontech), pDsRed-ER (Clontech Laboratories), and HA-Ubiquitin (Addgene).

To determine intracellular localization, cells grown on coverslips were cotransfected with a DHCR24 expression construct and an ER marker plasmid (pDsRed-ER, Clontech). Cells were then fixed with 3% (v/v) formaldehyde/PBS for 10 min. Cells were rinsed with PBS (three times for 5 min) and then permeabilized with 0.3% (v/v) Triton X-100/PBS for 5 min. Cells were washed with PBS, then incubated with 10% (v/v) FCS/PBS for 1 h. Cells were then incubated with 1:2000 V5 antibody in 10% FCS/PBS including 0.1% (w/v) saponin for 16 h at 4°C. After washing with 10% FCS/PBS (three times for 5 min), cells were incubated with 1:1,000 Alexa Fluor-488-conjugated secondary antibody (Molecular Probes) in 10% FCS/PBS for 1 h. Cells were washed with 10% FCS/PBS (three times for 5 min) and then mounted on glass slides with ProLong Gold AntiFade Reagent with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies). Images were obtained using a Nikon C1 confocal microscope with laser excitation at 408 nm (DAPI), 488 nm (DHCR24-V5) and 561 nm (ER marker).

T.C. Holmes (University of California, Irvine, CA) provided the rat Kv1.3 in a pRcCMV construct. The channel was subcloned into pEYFP-C1 and pECerulean-C1 (Clontech). All Kv1.3 mutants were generated in the pEYFP-Kv1.3 channel using a QuikChange site-directed mutagenesis kit (Agilent Technologies). pEYFP-Kv1.3CBD_less was subcloned into pECerulean-C1. For oocyte injection, Kv1.3 and Kv1.3CBD_less were subcloned into pcDNA3 and were placed under the control of a T7 promoter and then the cRNA was synthetized. J.R. Martens (University of Florida Medical School) provided the rat Caveolin 1 (Cav1) in pECerulean-C1. Cav1 was cloned into pcDNA3. The plasma membrane marker Akt-PH-pDsRed (pDsRed-tagged pleckstrin homology (PH) domain of Akt) was a kind gift of F. Viana (Universidad Miguel Hernández, Spain). The ER marker (pDsRed-ER) was obtained from Clontech. The mitochondrial marker (pmitoRFP) was constructed by fusing the mitochondrial transit sequence of the human isovaleryl coenzyme A dehydrogenase to the N-terminus of RFP (pDsRed1-N1, Clontech). Constructs were verified by sequencing.