Er STXBP1 is required for mast cell function. Surprisingly, we found

Er STXBP1 is required for mast cell function. Surprisingly, we found no significant functional discrepancy between wild-type and STXBP1-deficient mast cells both in vivo and in vitro suggesting that STXBP1 is dispensable for mast cell maturation and IgEdependent mast cell functions, and may point to functional redundancy in mast cell STXBPs.Reverse Transcription PCR (RT-PCR)Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed using SuperScript II Rnase H-Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. cDNA was used to amplify members of Sec/Munc (SM) family [14] with the following primers: STXBP1 forward, 59CCC GAG CAG CCA AAG TCA TC-39, and reverse, 59-GGT CTT CTC GCC AGT GTT CA-39, 605 bp; STXBP2 forward, MedChemExpress 86168-78-7 59-CAC TAT TAC ACG AAC TCA CG-39, and reverse, 59-ATG AGG CTG CTG TAC GAC T-39, 589 bp; STXBP3 forward, 59GGA AAA GAG AAG GAG GCA G-39, and reverse, 59-CCA AGG TGG CTC CAG TTA C-39, 515 bp; VPS33A forward, 59CGA GTT CCT GGA CAA GTG C-39, and reverse, 59-TGA GTT CTG GCT TCC TGT GA-39, 586 bp; VPS33B forward, 59-AGC TGG CTG GCA GAA TTA G-39, and reverse, 59-ATT GCG GAT CTC ACT GAA C-39, 566 bp; VPS45 forward, 59CTG AAC TTT GCC GAG ATT G-39, and reverse, 59-CTA ACC CGT TTA CCA CCA-39, 473 bp; SLY1 forward, 59-GGA TTC TGG GAA GAG TGG C-39, and reverse, 59-CAC AGG TGG CTT TTC AAC G-39, 442 bp; and actin forward, 59-ATC TGG CAC CAC ACC TTC TAC-39, and reverse, 59-GGA TGT CAA CGT CAC ACT TC-39, 612 bp. Each pair of primers spans at least one intron on genomic localization.JI 101 biological activity Materials and Methods AnimalsHeterozygous STXBP1 mice (STXBP1+/2) on a C57BL/6 background were purchased from Jackson Laboratory (http:// www.jax.org/). To minimize the effects of the genetic backgrounds, all mice were obtained by heterozygous mouse mating and littermate controls were used for all experiments. The protocols were approved by the University Committee on Laboratory Animals, Dalhousie University, in accordance with the guidelines of the Canadian Council on Animal Care.Enzyme-linked Immunosorbent Assay (ELISA) and Cytokine Multiplex AssayCytokines in the cell-free supernatants were determined using DuoSet ELISA kits obtained from R D Systems (Minneapolis, MN). Prostaglandin D2 (PGD2) enzyme immunoassay kit was obtained from Cayman (Ann Arbor, MI). Cell-free culture supernatants were also analyzed for cytokines using a Bio-Plex Pro mouse23-plex group 1 cytokine assay (Bio-Rad, Hercules, CA), which detects 23 cytokines and chemokines. The assay was performed according to the manufacturer’s protocol and was read on a Bio-Plex 200 HTF multiplex array system. Data were analyzed using Bio-Plex Manager version 6.0 software (Bio-Rad).AntibodiesAntibodies to phospho-JNK (Thr-183/Tyr-185), JNK, phospho-p38 MAPK (Thr-180/Tyr-182), phospho-p44/42 (ERK1/2), p44/42 MAPK, phospho-IkB-a (Ser 32), IkB-a, phospho-Akt (Ser 473), Akt, STXBP1, and PKG-1 were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies to p38 MAPK and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to syntaxin-1 was purchased from Sigma (St. Louis, MO). FITC-conjugated rat anti-mouse IgE (IgG1), FITC-rat IgG1 and FITC-conjugated rat anti-mouse CD117 (c-kit) were purchased from Cedarlane Laboratories (Burlington, ON, Canada).Western BlottingCells were lysed in radioimmunoprecipitation (RIPA) assay buffer supplemented with a panel of protease and phosphatase inhibitors. Cleared lysates (35 mg protein.Er STXBP1 is required for mast cell function. Surprisingly, we found no significant functional discrepancy between wild-type and STXBP1-deficient mast cells both in vivo and in vitro suggesting that STXBP1 is dispensable for mast cell maturation and IgEdependent mast cell functions, and may point to functional redundancy in mast cell STXBPs.Reverse Transcription PCR (RT-PCR)Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed using SuperScript II Rnase H-Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. cDNA was used to amplify members of Sec/Munc (SM) family [14] with the following primers: STXBP1 forward, 59CCC GAG CAG CCA AAG TCA TC-39, and reverse, 59-GGT CTT CTC GCC AGT GTT CA-39, 605 bp; STXBP2 forward, 59-CAC TAT TAC ACG AAC TCA CG-39, and reverse, 59-ATG AGG CTG CTG TAC GAC T-39, 589 bp; STXBP3 forward, 59GGA AAA GAG AAG GAG GCA G-39, and reverse, 59-CCA AGG TGG CTC CAG TTA C-39, 515 bp; VPS33A forward, 59CGA GTT CCT GGA CAA GTG C-39, and reverse, 59-TGA GTT CTG GCT TCC TGT GA-39, 586 bp; VPS33B forward, 59-AGC TGG CTG GCA GAA TTA G-39, and reverse, 59-ATT GCG GAT CTC ACT GAA C-39, 566 bp; VPS45 forward, 59CTG AAC TTT GCC GAG ATT G-39, and reverse, 59-CTA ACC CGT TTA CCA CCA-39, 473 bp; SLY1 forward, 59-GGA TTC TGG GAA GAG TGG C-39, and reverse, 59-CAC AGG TGG CTT TTC AAC G-39, 442 bp; and actin forward, 59-ATC TGG CAC CAC ACC TTC TAC-39, and reverse, 59-GGA TGT CAA CGT CAC ACT TC-39, 612 bp. Each pair of primers spans at least one intron on genomic localization.Materials and Methods AnimalsHeterozygous STXBP1 mice (STXBP1+/2) on a C57BL/6 background were purchased from Jackson Laboratory (http:// www.jax.org/). To minimize the effects of the genetic backgrounds, all mice were obtained by heterozygous mouse mating and littermate controls were used for all experiments. The protocols were approved by the University Committee on Laboratory Animals, Dalhousie University, in accordance with the guidelines of the Canadian Council on Animal Care.Enzyme-linked Immunosorbent Assay (ELISA) and Cytokine Multiplex AssayCytokines in the cell-free supernatants were determined using DuoSet ELISA kits obtained from R D Systems (Minneapolis, MN). Prostaglandin D2 (PGD2) enzyme immunoassay kit was obtained from Cayman (Ann Arbor, MI). Cell-free culture supernatants were also analyzed for cytokines using a Bio-Plex Pro mouse23-plex group 1 cytokine assay (Bio-Rad, Hercules, CA), which detects 23 cytokines and chemokines. The assay was performed according to the manufacturer’s protocol and was read on a Bio-Plex 200 HTF multiplex array system. Data were analyzed using Bio-Plex Manager version 6.0 software (Bio-Rad).AntibodiesAntibodies to phospho-JNK (Thr-183/Tyr-185), JNK, phospho-p38 MAPK (Thr-180/Tyr-182), phospho-p44/42 (ERK1/2), p44/42 MAPK, phospho-IkB-a (Ser 32), IkB-a, phospho-Akt (Ser 473), Akt, STXBP1, and PKG-1 were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies to p38 MAPK and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to syntaxin-1 was purchased from Sigma (St. Louis, MO). FITC-conjugated rat anti-mouse IgE (IgG1), FITC-rat IgG1 and FITC-conjugated rat anti-mouse CD117 (c-kit) were purchased from Cedarlane Laboratories (Burlington, ON, Canada).Western BlottingCells were lysed in radioimmunoprecipitation (RIPA) assay buffer supplemented with a panel of protease and phosphatase inhibitors. Cleared lysates (35 mg protein.

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