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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Mitochondrion. 2013 Feb 21;13(6):10.1016/j.mito.2013.02.002. doi: 10.1016/j.mito.2013.02.002

Fig. 4. Mammalian SIRT4 specifically interacts with mitochondrial biotin-dependent carboxylases.

Fig. 4

(A) Expression vectors encoding SIR-2.2-GFP, SIR-2.3-GFP or GFP alone were cotransfected in HEK293 cells with expression vectors for either FLAG-tagged C. elegans PCCA-1, MCCC-1 or PYC-1 or mouse ACDH-3 or HP1β. GFP-tagged proteins were immunoprecipitated with GFP-TrapA and analyzed by SDS-PAGE and Western blotting using anti-FLAG, anti-SIR-2.2 and anti-GFP antibodies. I: input (2%); S: supernatant after immunoprecipitation (2%); IP: immunoprecipitated material. (B) Similar analysis as in (A) but using vectors encoding mouse SIRT4-GFP, PCCA-FLAG, MCCC1-FLAG and PC-FLAG as well as GFP. (C) Expression vectors for human SIRT3, SIRT4, SIRT5 and CDYL1c (MYC-tagged) were cotransfected with expression vectors encoding either murine PC, MCCC1, PCCA of HP1β (FLAG-tagged) in HEK293 cells. Proteins were immunoprecipitated with anti-FLAG antibodies and analyzed by Western blotting with anti-MYC and anti-FLAG antibodies (top). Cell extracts (input) were directly analyzed in Western blotting with anti-MYC antibodies to determine expression levels of tagged protein in each experiment (bottom).