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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 3. — Human Parkin Ser⁶⁵ is a substrate of human PINK1 upon CCCP stimulation. (a) Confirmation by mass spectrometry that Ser⁶⁵ of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser¹³¹ and Ser⁶⁵ phosphopeptide (3⁺ R.NDWTVQNCDLDQQSIVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y-axis and retention time is plotted on the x-axis. The m/z value corresponding to the Ser¹³¹ phosphopeptide was detected in all conditions whilst that of the Ser⁶⁵ phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. (b) Characterization of Parkin phospho-Ser⁶⁵ antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive 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 for 3 h. 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. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.