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

Figure 7.

Figure 7.

Sas10 protects Mpp10 from Capn3-mediated degradation and determines the nucleolar-localization of Mpp10. (A) Prediction of a Capn3-recognition motif (211MEAFLDDMD) at the N-terminal of Mpp10 based on the Capn3 recognition consensus sequence. The MEAFLDDMD motif was deleted in Myc-tagged m0 (Myc-m0-x) and m2 (Myc-m2-x). (B) Myc-m0, Myc-m0-x, Myc-m2 or Myc-m2-x was respectively transfected into 293T cells together with or without HA-Sas10 (HA-s0) plasmid. Western blot was performed to compare the stability of corresponding proteins. (C and D) Protein samples were extracted from 293T cells expressing, respectively, Capn3, Myc-m0, Myc-m0-x, Myc-m2 or Myc-m2-x. Capn3 protein extract was mixed with Myc-m0 or Myc-m0-x (C) and with Myc-m2 or Myc-m2-x (D) with or without EDTA for 2 and 5 min. Western blot was performed to compare the protein stability in each reaction. (E) Enzymatic activity assays using purified His-Capn3 or His-Capn3C129S (C129S) as the enzyme and purified His-Mpp10 or His-Mpp10-X as the substrate. Reaction mixture containing 10 mM Ca2+ or 20 mM EDTA was incubated at 37°C for 5 and 15 min as indicated. (F) Myc-mpp10 was transfected into 293T cells alone or together with HA-sas10 or HA-sas10-s5 (lacking the Mpp10-interacting domain) plasmid. The cells were subjected to co-immunostaining of Myc-Mpp10 and Fib, Myc-Mpp10 and HA-Sas10, or Myc-Mpp10 and HA-Sas10-s5 48 h post-transfection; bar, 10 μm.