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. 2019 Feb 18;47(6):2996–3012. doi: 10.1093/nar/gkz105

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — Stabilities of Sas10 and Mpp10 are interdependent. (A) The stabilities of Sas10 and Mpp10 mutually depend on each other in a dosage-dependent manner. Lanes 1–3: HA-Sas10 plasmid (500 ng) was co-transfected with different amount of Myc-Mpp10 plasmid. Lanes 4–6: Myc-Mpp10 plasmid (500 ng) was co-transfected with different amount of HA-Sas10 plasmid. : 0 ng, 250 ng, 500 ng. The pCS2⁺ plasmid was used to make up the total amount of 500 ng plasmid DNA for transfection. Lane 7: control cells transfected with the DsRed plasmid. Mpp10 and Sas10 were detected using their specific antibodies, respectively. Total proteins were extracted from 293T cells 48 h post-transfection. (B) Western blot of Mpp10, Sas10, Fib and α-Tub in sas10^Δ2 and mpp10^Δ10 homozygous mutants and their siblings (WT and heterozygous). Total proteins were extracted from 5dpf-old embryos. (C–E) In vitro assay of Myc-Mpp10 degradation by Capn3. Capn3, Myc-Mpp10 or Myc-Mpp10 together with HA-Sas10 was respectively expressed in 293T cells. Protein extracts were mixed as indicated (C and D) and the mixture was incubated in the reaction buffer containing 3 mM CaCl₂ at 37°C for different time intervals (min) as shown. The ratio of Myc-Mpp10 at different time point against Myc-Mpp10^{0 min} in Capn3+Myc-Mpp10 or in Capn3+Mpp10+ HA-Sas10 with or without EDTA reaction mixture was plotted against the time interval (E). α-Tub: loading control.

Inline graphic — Stabilities of Sas10 and Mpp10 are interdependent. (A) The stabilities of Sas10 and Mpp10 mutually depend on each other in a dosage-dependent manner. Lanes 1–3: HA-Sas10 plasmid (500 ng) was co-transfected with different amount of Myc-Mpp10 plasmid. Lanes 4–6: Myc-Mpp10 plasmid (500 ng) was co-transfected with different amount of HA-Sas10 plasmid. : 0 ng, 250 ng, 500 ng. The pCS2⁺ plasmid was used to make up the total amount of 500 ng plasmid DNA for transfection. Lane 7: control cells transfected with the DsRed plasmid. Mpp10 and Sas10 were detected using their specific antibodies, respectively. Total proteins were extracted from 293T cells 48 h post-transfection. (B) Western blot of Mpp10, Sas10, Fib and α-Tub in sas10^Δ2 and mpp10^Δ10 homozygous mutants and their siblings (WT and heterozygous). Total proteins were extracted from 5dpf-old embryos. (C–E) In vitro assay of Myc-Mpp10 degradation by Capn3. Capn3, Myc-Mpp10 or Myc-Mpp10 together with HA-Sas10 was respectively expressed in 293T cells. Protein extracts were mixed as indicated (C and D) and the mixture was incubated in the reaction buffer containing 3 mM CaCl₂ at 37°C for different time intervals (min) as shown. The ratio of Myc-Mpp10 at different time point against Myc-Mpp10^{0 min} in Capn3+Myc-Mpp10 or in Capn3+Mpp10+ HA-Sas10 with or without EDTA reaction mixture was plotted against the time interval (E). α-Tub: loading control.