Reptavidin Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 hours

Reptavidin Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 hours at 4uC, and the remaining supernatant was kept as the input. The beads were subsequently washed five times with 16 lysis buffer before elution with 50 ul of 26 NuPAGE sample buffer (Invitrogen, Carlsbad, California, USA) plus 100 mM DTT at 37uC for 10 minutes. These biotinylated fractions were analyzed as TRPM4 expressed at the cell surface. The input fractions, analyzed as total expression of TRPM4, were resuspended with 46 NuPAGE Sample Buffer plus 100 mM DTT to give a concentration of 1 mg/ml and incubated at 37uC for 10 minutes.Preparation of TRPM4 MutantsThe complete human wild-type TRPM4 cDNA was cloned in pcDNA4/TO vector (Invitrogen, Cergy Pontoise, France) [16]. Mutants were obtained by in vitro mutagenesis using QuickChange II site-directed mutagenesis kit (Agilent Technologies, Massy, France). Mutant cDNA clones were systematically resequenced before use in further experiments.Stable TRPM4 Mutant ExpressionpcDNA4/TO plasmid containing the diverse TRPM4 mutants were used to transfect T-RExTM 293 cell lines with Lipofectamine 2000 (Invitrogen, Cergy Pontoise, France) according to manufacturer specifications. The T-RExTM 293 cell line 307538-42-7 stably expresses the tetracycline repressor protein enabling the silencing of the gene of interest unless tetracycline is added to the culture medium. TRExTM 293 is a stable transformed cell line of HEK 293 obtained with a plasmid that encodes the Tet repressor under the control of the human CMV promoter. Several stable clones (3?) of each TRPM4 mutant were obtained according to Invitrogen protocol by selecting with blasticidin (Tet repressor) and zeocin (TRPM4). These stable clones were used for the electrophysiological study.Western Blotting ElectrophysiologyCurrents were recorded from whole-cell or inside-out patches of T-RexTM 293 transfected cells with a patch-clamp amplifier Axopatch 200B (Axon instruments, Forster city, CA, USA) using pClamp 9 software (Axon instruments). Experiments were conducted at room temperature. For patch-clamp experiments in inside-out conditions, cells were bathed in a solution containing (in mM): 140 NaCl; 4.8 KCl; 1.2 MgCl2; 0.1 CaCl2; 10 glucose; and 10 HEPES, pH 7.4 (with NaOH). Solutions perfused at the inside of the membrane contained the previous solution (with 1 mM CaCl2) or, for MedChemExpress Tubastatin-A determination of ionic selectivity, a low NaCl solution (in mM):Both input and biotinylated fractions were analyzed on 8 polyacrylamide gel and detected with anti-TRPM4 antibody raised against the C terminal portion of TRPM4 from amino-acids 1138 to 1156 (Pineda, Berlin, Germany) and anti-a-actin A2066 (Sigma, St. Louis, Missouri, USA) antibodies. The blots obtained were quantified using IGOR Pro (Wavemetrics, Lake Oswego, Oregon, USA) software.StatisticsVariant prevalence in the BrS vs control cohorts was tested by the Fisher exact test and one sided p values are presented in table 1. Mutant electrophysiological values and quantified bands onTable 1. Presentation of TRPM4 variants.mRNA 10457188 101 58 125 ?mammals except rodent 0/7 N-term. Intracyto 0/2000 0/5366 0/3501 0/7366 0.0323* mammals 4/7 N-term. Intracyto 0/2000 0/5356 0/3495 0/7356 0.0326* ?vertebrates 7/7 N-term. Intracyto 0/300 0/3501 0/1864 0/3801 0.0612 ?mammals 0/7 N-term. Intracyto 0/2040 0/3490 0/1854 0/5530 0.0429* 0/7384 0/5665 0/ProteinGrantham [0-215] Splicing Controls Total 1 TotalInterspecies InterTRPM conservation conservation P.Reptavidin Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 hours at 4uC, and the remaining supernatant was kept as the input. The beads were subsequently washed five times with 16 lysis buffer before elution with 50 ul of 26 NuPAGE sample buffer (Invitrogen, Carlsbad, California, USA) plus 100 mM DTT at 37uC for 10 minutes. These biotinylated fractions were analyzed as TRPM4 expressed at the cell surface. The input fractions, analyzed as total expression of TRPM4, were resuspended with 46 NuPAGE Sample Buffer plus 100 mM DTT to give a concentration of 1 mg/ml and incubated at 37uC for 10 minutes.Preparation of TRPM4 MutantsThe complete human wild-type TRPM4 cDNA was cloned in pcDNA4/TO vector (Invitrogen, Cergy Pontoise, France) [16]. Mutants were obtained by in vitro mutagenesis using QuickChange II site-directed mutagenesis kit (Agilent Technologies, Massy, France). Mutant cDNA clones were systematically resequenced before use in further experiments.Stable TRPM4 Mutant ExpressionpcDNA4/TO plasmid containing the diverse TRPM4 mutants were used to transfect T-RExTM 293 cell lines with Lipofectamine 2000 (Invitrogen, Cergy Pontoise, France) according to manufacturer specifications. The T-RExTM 293 cell line stably expresses the tetracycline repressor protein enabling the silencing of the gene of interest unless tetracycline is added to the culture medium. TRExTM 293 is a stable transformed cell line of HEK 293 obtained with a plasmid that encodes the Tet repressor under the control of the human CMV promoter. Several stable clones (3?) of each TRPM4 mutant were obtained according to Invitrogen protocol by selecting with blasticidin (Tet repressor) and zeocin (TRPM4). These stable clones were used for the electrophysiological study.Western Blotting ElectrophysiologyCurrents were recorded from whole-cell or inside-out patches of T-RexTM 293 transfected cells with a patch-clamp amplifier Axopatch 200B (Axon instruments, Forster city, CA, USA) using pClamp 9 software (Axon instruments). Experiments were conducted at room temperature. For patch-clamp experiments in inside-out conditions, cells were bathed in a solution containing (in mM): 140 NaCl; 4.8 KCl; 1.2 MgCl2; 0.1 CaCl2; 10 glucose; and 10 HEPES, pH 7.4 (with NaOH). Solutions perfused at the inside of the membrane contained the previous solution (with 1 mM CaCl2) or, for determination of ionic selectivity, a low NaCl solution (in mM):Both input and biotinylated fractions were analyzed on 8 polyacrylamide gel and detected with anti-TRPM4 antibody raised against the C terminal portion of TRPM4 from amino-acids 1138 to 1156 (Pineda, Berlin, Germany) and anti-a-actin A2066 (Sigma, St. Louis, Missouri, USA) antibodies. The blots obtained were quantified using IGOR Pro (Wavemetrics, Lake Oswego, Oregon, USA) software.StatisticsVariant prevalence in the BrS vs control cohorts was tested by the Fisher exact test and one sided p values are presented in table 1. Mutant electrophysiological values and quantified bands onTable 1. Presentation of TRPM4 variants.mRNA 10457188 101 58 125 ?mammals except rodent 0/7 N-term. Intracyto 0/2000 0/5366 0/3501 0/7366 0.0323* mammals 4/7 N-term. Intracyto 0/2000 0/5356 0/3495 0/7356 0.0326* ?vertebrates 7/7 N-term. Intracyto 0/300 0/3501 0/1864 0/3801 0.0612 ?mammals 0/7 N-term. Intracyto 0/2040 0/3490 0/1854 0/5530 0.0429* 0/7384 0/5665 0/ProteinGrantham [0-215] Splicing Controls Total 1 TotalInterspecies InterTRPM conservation conservation P.

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