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Immunoprecipitation and western blot show interaction of otoferlin with CaMKIIδ.？(A？C)？Two HA-tagged mouse otoferlin fragments, C2ABC (aa 1？632 in？NP_001093865; 70 kDa) and C2DEF (aa 933？1920; 114 kDa) were co-transfected with mcherry-tagged mouse CaMKIIδ into HEK293 cells. Transfections were performed either with otoferlin C2ABC and CaMKIIδ (A, Input Lane 1 and 2), otoferlin C2DEF and CaMKIIδ (B, Input Lane 1 and 2) or in the presence of both C2ABC and C2DEF fragments and CaMKIIδ (C, Input Lane 1 and 2). Co-immunoprecipitations of C2ABC-HA and C2DEF-HA were conducted from HEK293 cell lysates using anti-HA antibodies (p/n 600-401-384). CaMKIIδ-mcherry was detected in the eluate using an anti-RFP (red fluorescent protein) antibody (p/n 200-301-379) (A？C, Lane 3), indicating that CaMKIIδ co-precipitated with recombinant otoferlin fragments. Secondary anti-rabbit Dylight680 (p/n 611-144-003) and anti-mouse Dylight800 antibodies (610-145-003) (1:10,000). FIGURE 5. PMID: 29046633.
C.？Representative western blots, original blots are shown in？(supplementary Fig S8-9). And densitometric quantification of relative protein levels from western blots. Data are depicted as mean？±？SD, n？=？3, **P？<？0.01, ***P？<？0.001 and ****P？<？0.0001 by one-way ANOVA. Intracellular transport, activation, mitochondrial transport, β-oxidation, carnitine shuttle, and auxiliary proteins. The primary antibodies used as follows: VLCAD 1:1000, MCAD 1:1000, LCAD 1:1000, TFPa 1:500, TFPb 1:3000, CPT1α 1:1000, and GAPDH 1:30,000 dilutions overnight at 4？°C. The membranes were then incubated with fluorescent conjugated secondary antibodies for 1？h; DyLight 800 conjugated goat Anti-Rabbit IgG (611-145-002), DyLight 680 conjugated goat Anti-Rabbit IgG (611-144-003), DyLight 800 conjugated goat Anti-Mouse IgG (610-145-002), and DyLight 680 conjugated donkey Anti-Mouse IgG (610-744-124). Fig 1. PMID: 33725513.
Assessment of mitochondrial fusion and fission.B.？Representative western blots (original blots are shown in？supplementary Fig. S10) and quantification of MFN1/2 and DRP1. No significant changes in the relative levels of proteins that facilitate mitochondrial fusion (MFN1/2) and fission (DRP1) between non-disease (control) and mutant primary fibroblasts. Data are depicted as mean？±？SD, n？=？3. The primary antibodies used as follows: MFN1 1:400, MFN2 ( 1:400, DRP1 1:100 and GAPDH 1:30,000 dilutions overnight at 4？°C. The membranes were then incubated with fluorescent conjugated secondary antibodies for 1？h; DyLight 800 conjugated goat Anti-Rabbit IgG (611-145-002), Antibody DyLight 680 conjugated Anti-Rabbit IgG made in goat (611-144-003), DyLight 800 conjugated goat Anti-Mouse IgG (610-145-002), and DyLight 680 conjugated donkey Anti-Mouse IgG (610-744-124). Fig 3. PMID: 33725513.
Properties of DyLight？ Conjugates.
DyLight？ dyes can be used for two-color Western Blot detection with low background and high signal.？ Anti-tubulin was detected using a DyLight？ 680 conjugate.？ Anti-TNFa was detected using a DyLight？ 800 conjugate. The image was captured using the OdysseyR Infrared Imaging System developed by LI-COR.
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Immunoprecipitation and western blot show interaction of otoferlin with CaMKIIδ.？(A？C)？Two HA-tagged mouse otoferlin fragments, C2ABC (aa 1？632 in？NP_001093865; 70 kDa) and C2DEF (aa 933？1920; 114 kDa) were co-transfected with mcherry-tagged mouse CaMKIIδ into HEK293 cells. Transfections were performed either with otoferlin C2ABC and CaMKIIδ (A, Input Lane 1 and 2), otoferlin C2DEF and CaMKIIδ (B, Input Lane 1 and 2) or in the presence of both C2ABC and C2DEF fragments and CaMKIIδ (C, Input Lane 1 and 2). Co-immunoprecipitations of C2ABC-HA and C2DEF-HA were conducted from HEK293 cell lysates using anti-HA antibodies (p/n 600-401-384). CaMKIIδ-mcherry was detected in the eluate using an anti-RFP (red fluorescent protein) antibody (p/n 200-301-379) (A？C, Lane 3), indicating that CaMKIIδ co-precipitated with recombinant otoferlin fragments. Secondary anti-rabbit Dylight680 (p/n 611-144-003) and anti-mouse Dylight800 antibodies (610-145-003) (1:10,000). FIGURE 5. PMID: 29046633.
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| 別品名 |
Goat Anti Rabbit IgG F(c) DyLight 680TM Conjugated Antibody, Goat Anti-Rabbit IgG Fc Fragment Antibody DyLight 680TM conjugation, Goat Anti Rabbit IgG Fc Antibody DyLight 680TM conjugated
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| 交差種 |
Rabbit
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| 免疫動物 |
Goat
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| 標識物 |
DyLightTM 680
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| 精製度 |
Affinity Purified
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| 参考文献 |
[Pub Med ID]29046633
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| [注意事項] |
濃度はロットによって異なる可能性があります。メーカーDS及びCoAからご確認ください。
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| メーカー |
品番 |
包装 |
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RKL
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611-144-003
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100 UG
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※表示価格について
| 当社在庫 |
なし
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| 納期目安 |
約10日
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| 法規制 |
毒
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| 保存温度 |
４℃
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