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. 2012 May;2(5):120080. doi: 10.1098/rsob.120080

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© 2012 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 7. — Timecourse of CCCP-induced activation of PINK1. (a) Timecourse of PINK1 autophosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing PINK1-FLAG wild-type and kinase-inactive (D384A) were stimulated at the indicated time points with 10 μM of CCCP. 0.5 mg of mitochondrial extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with anti-phospho-Thr²⁷⁵ antibody or total PINK1 antibody. (b) No time-dependent activation of cytoplasmic PINK1 in vivo. As in (a) cytoplasmic extracts were obtained at the indicated time-points and immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with PINK1 anti-phospho-Thr²⁷⁵ antibody or total PINK1 antibody. (c) Timecourse of Parkin Ser⁶⁵ phosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing wild-type PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser⁶⁵Ala (S65A) mutant Parkin; induced with doxycycline and stimulated with 10 μM of CCCP at the indicated time points. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser⁶⁵ antibody in the presence of dephosphorylated peptide. One per cent of the IP was immunoblotted with total anti-Parkin antibody. 1.5 mg of whole-cell extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody.