mouse bk channel  (Addgene inc)

Journal: Cardiovascular Research

Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

doi: 10.1093/cvr/cvaa089

Figure Lengend Snippet: Knockdown of RyR1/RyR2 reduced the interaction of BKCa channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).

Article Snippet: Uterine arterial slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature (RT) for 1 h, and then incubated with primary antibodies, rabbit polyclonal MaxiKβ antibody (1:200; sc-33608, Santa Cruz Biotechnology), rabbit polyclonal RyR1 antibody (1:100; 8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (1:100; ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (1:100; AB9082, EMD Millipore), mouse monoclonal BK Ca channel α (1:200; sc-374142), or mouse monoclonal BK Ca channel β1 (1:200; sc-377023) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence, Staining

Journal: Cardiovascular Research

Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

doi: 10.1093/cvr/cvaa089

Figure Lengend Snippet: Knockdown of RyR1/RyR2 downregulated the expression of BKCa channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BKCa channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

Article Snippet: Uterine arterial slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature (RT) for 1 h, and then incubated with primary antibodies, rabbit polyclonal MaxiKβ antibody (1:200; sc-33608, Santa Cruz Biotechnology), rabbit polyclonal RyR1 antibody (1:100; 8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (1:100; ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (1:100; AB9082, EMD Millipore), mouse monoclonal BK Ca channel α (1:200; sc-374142), or mouse monoclonal BK Ca channel β1 (1:200; sc-377023) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Cardiovascular Research

Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

doi: 10.1093/cvr/cvaa089

Figure Lengend Snippet: Pregnancy increased co-localization of RyR1/RyR2 with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and RyR1, RyR2, or RyR3 (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.

Article Snippet: Uterine arterial slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature (RT) for 1 h, and then incubated with primary antibodies, rabbit polyclonal MaxiKβ antibody (1:200; sc-33608, Santa Cruz Biotechnology), rabbit polyclonal RyR1 antibody (1:100; 8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (1:100; ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (1:100; AB9082, EMD Millipore), mouse monoclonal BK Ca channel α (1:200; sc-374142), or mouse monoclonal BK Ca channel β1 (1:200; sc-377023) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight.

Techniques: Immunofluorescence, Staining

Journal: Cardiovascular Research

Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

doi: 10.1093/cvr/cvaa089

Figure Lengend Snippet: Knockdown of RyR1/RyR2 reduced the interaction of BKCa channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).

Article Snippet: Uterine arterial slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature (RT) for 1 h, and then incubated with primary antibodies, rabbit polyclonal MaxiKβ antibody (1:200; sc-33608, Santa Cruz Biotechnology), rabbit polyclonal RyR1 antibody (1:100; 8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (1:100; ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (1:100; AB9082, EMD Millipore), mouse monoclonal BK Ca channel α (1:200; sc-374142), or mouse monoclonal BK Ca channel β1 (1:200; sc-377023) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence, Staining

Journal: Cardiovascular Research

Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

doi: 10.1093/cvr/cvaa089

Figure Lengend Snippet: Knockdown of RyR1/RyR2 downregulated the expression of BKCa channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BKCa channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

Article Snippet: Uterine arterial slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature (RT) for 1 h, and then incubated with primary antibodies, rabbit polyclonal MaxiKβ antibody (1:200; sc-33608, Santa Cruz Biotechnology), rabbit polyclonal RyR1 antibody (1:100; 8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (1:100; ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (1:100; AB9082, EMD Millipore), mouse monoclonal BK Ca channel α (1:200; sc-374142), or mouse monoclonal BK Ca channel β1 (1:200; sc-377023) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight.

Techniques: Expressing, Western Blot