SUPPLEMENTARY INFORMATION FIGURE 1-1
SUPPLEMENTARY INFORMATION FIGURE 2-2
SUPPLEMENTARY INFORMATION METHODS GST-Pull-Down. Cultures of E. Coli (BL21) were transformed with pgex (Clontech) and pgex recombinant vectors to produce GST and GST-TWIK1 proteins. These proteins were purified on gluthation-sepharose beads. Twenty-four hours after transfection with the EFA6GFP, or ARF6T27Nmyc construct, or both, BHK 21 cells (5.10 6 per condition) were lysed at 4 C in 1 ml lysis buffer [50 mmtris-hcl, ph8.0; 100mM NaCl; 10 mm MgCl 2 ; 1% Triton X-100; 0.005% SDS; 10% glycerol; 2mM DTT with protease inhibitors (Roche Diagnostics)]. Lysates were clarified by centrifugation at 13,000 g for 10 minutes and incubated with 0.5% bovine serum albumin and 30 µg GST-TWIK or 40 µg GST bound to glutathione-sepharose beads (Amersham Biosciences) for 45 minutes. The beads were washed three times in lysis buffer. Bound proteins were eluted by boiling in 30 µl SDS-PAGE sample buffer, separated by SDS-PAGE and blotted onto cellulose (Hybond C, Amersham Biosciences). Positive bands were revealed using the supersignal WestPico luminescent detection system (Pierce Biotechnology, Rockford, IL, USA). Cell culture, reagents and antibodies. BHK 21 cells were grown in BHK21 medium (Invitrogen, Cergy Pontoise, F) supplemented with 5% foetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, 10% tryptose, 2 mm L-Glutamine in incubators with 5% CO 2 at 37 C. MDCK cells were grown in Minimal Essentiel Medium (MEM, Invitrogen) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin in humidified incubators with 5% CO 2 at 37 C. For polarization, MDCK cells were grown 5 days on 12- mm, 0.4 µm pore size Transwell polycarbonate filters (Corning Costar, Cambridge, MA, USA). HeLa (Henrietta Lacks) cells were grown in Dulbecco s Modified Essential Medium (DMEM, Invitrogen) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin in humidified incubators with 5% CO 2 at 37 C. - 3
The following antibodies were used: rat monoclonal antibody (mabs) against hemagglutinin (HA) epitope (clone 3F10, Roche Diagnostics, Mannheim, Germany), mouse monoclonal antibody (mab) against Myc epitope (clone 9E10, Santa Cruz Biotchenology, Santa Cruz, Ca, USA), rabbit polyclonal anti-twik-αe and -αc antibodies (Lesage F, Reyes R, Fink M, Duprat F, Guillemare E and Lazdunski M (1996) Dimerization of TWIK-1 K + channel subunits via a disulfide bridge. Embo J 15: 6400-6407), rabbit anti-gfp antibody (Clontech), mouse monoclonal anti-α-tubulin (clone Tub 2.1, Sigma, Saint Louis, USA). Nuclei were labeled with Hoechst 33342 (Molecular Probes Europe BV, Leiden, N) and polymerized actin with Alexa Fluor 488 phalloidin (Molecular Probes). Transferrin-FITC was from Sigma. Texas Red and Cy5 conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Peroxidase conjugated antibodies were from Amersham Biosciences. Generation of stable MDCK cell line. MDCK cells were co-transfected with pci-twik1 or phcredc1-twik1 and pires-neo (Clontech) vectors using Lipofectamine 2000. Stably transfected clones were selected in a medium containing neomycin (1 mg/ml). Immunofluorescence and transferrin internalization. BHK21 cells were plated on 12-mm glass coverslips before transfection. Cells were transfected with 1 µg of pci-ires-cd8- TWIK-1 and 0.5 µg EFA6-GFP or 1 µg of ARF6T27Nmyc constructs or both using Fugene 6 transfection reagent described by the manufacturer (Roche Diagnostics). Twenty-four hours after transfection, cells were fixed for 20 minutes with a 4% (w/v) paraformaldehyde/pbs and processed for immunofluorescence analysis as previously described (Franco M, Boretto J, Robineau S, Monier S, Goud B, Chardin P and Chavrier P (1998) ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function. Proc Natl Acad Sci U S A 95: 9926-9931). Briefly, cells were permeabilized with 0.5% saponin and blocked with 10% horse serum in phosphate saline - 4
buffer; primary and secondary antibodies were diluted in 5% horse serum/pbs and incubated 1 hour at room temperature. Coverslips were then mounted in Dako Fluorescent Mounting medium (Dako Corporation, Carpinteria, CA, USA). MDCK and stable MDCK for TWIK1 cells were also plated on 12-mm glass coverslips and transfected or not with HcRedTWIK1 or Vamp8-EGFP using Lipofectamine 2000 (Invitrogen). Cells were fixed with a 4% (w/v) paraformaldehyde solution or treated with nocodazole (10 µm during 30 minutes at 37 C) before fixation and processed for immunofluorescence analysis. Polarized MDCK cells expressing TWIK1 were fixed and processed for immunofluorescence as described previously. HeLa cells were plated on 12-mm glass coverslips before transfection with TWIK1, TWIK1C69S and TWIK1ΔCter using Fugene 6 method. Twenty-four hours after transfection, HeLa cells were washed twice in PBS (ph 7.4) and incubated in serum free DMEM containing 0.1 mg/ml Bovin Serum Albumin (BSA, Sigma) for 2 hours at 37 C. Cells were treated with transferrin-fitc (25 µg/ml in serum free DMEM containing 0.1 mg/ml BSA) for the indicated amount of time, then fixed in 4% paraformaldehyde and processed for immunofluorescence. Microscopy analysis was carried out with a Leica TCS-SP microscope equipped with a mixed-gas argon/krypton laser (Leica Microsytems, Reuil Malmaison, F) or with Axioplan 2 Imaging microscope equipped with a high pressure mercury lamp (Carl Zeiss, Le Pecq, F). - 5