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. 2017 Oct 1;130(19):3248–3260. doi: 10.1242/jcs.201400

Fig. 2.

Fig. 2.

Engineering of APEX2 constructs of Mic60 and Sam50. (A) Clone and linker peptide length between the C-terminal end of Mic60 or Sam50 and APEX2. Only Mic60 with the 24-residue linker and Sam50 with the 48-residue linker showed expression. (B) Partial structure prediction of the C-terminal region of Mic60 and Sam50 rendered with the structure of APEX2 attached. (C) Flag-tagged Mic19 along with APEX2-tagged Mic60 and Sam50 were transiently expressed in HEK 293 cells and immunoprecipitated (IP) using FLAG resin. The eluted samples were analyzed by immunoblotting with antibodies against Flag, Mic19, Mic60 and Sam50. Mic19 interacted with both the endogenous and the APEX2-tagged MIc60 and Sam50. A 10% input control is shown on the right. The lower molecular mass band represents endogenous protein. (D) Bright-field light microscopy of Mic60–APEX2- and Sam50–APEX2-transfected human primary astrocytes showing mitochondrial labeling. Results are representative of n=10 experiments.