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. 2015 Sep 30;35(21):3768–3784. doi: 10.1128/MCB.00184-15

FIG 3.

FIG 3

cPLA2α inhibits SIRT2 deacetylase activity. (A) LLC-mCherry cells (lanes 1 and 3) and LLC-SIRT2-mCherry cells (lanes 2 and 4) were infected with Ad-LacZ or Ad-cPLA2α. Levels of Ac-H4K16 were examined by Western blotting in total lysates. Levels of SIRT2, cPLA2α, ERK, and H4K16 were compared in total lysates. Intensities of Ac-H4K16 bands were quantified by ImageJ from three independent experiments. (B) WT and cPLA2α−/− MEFs were infected with Ad-LacZ or Ad-cPLA2α. Levels of Ac-H4K16 were detected by Western blotting in total lysates. Intensities of Ac-H4K16 bands were quantified by ImageJ (n = 3). (C) LLC-PK1 cells were mock transfected (lane 1), transfected with SIRT2 (lane 2), or cotransfected with SIRT2 and WT cPLA2α (lane 3) or with SIRT2 and cPLA2αS228A (lane 4). Cell lysates were subjected to Western blotting for detection of Ac-H4K16. Intensities of Ac-H4K16 bands were quantified by ImageJ (n = 3). (D) LLC-PK1 cells were transfected with SIRT2, SIRT2 and cPLA2α, or SIRT2 and cPLA2αΔN215. Ac-H4K16 levels were quantified by Western blotting. Intensities of Ac-H4K16 bands were quantified by ImageJ (n = 3). (E) SIRT2 was immunoprecipitated from mock-transfected LLC-PK1 cells (no SIRT2), SIRT2-transfected cells (SIRT2), or LLC-PK1 cells cotransfected with SIRT2 and cPLA2α (SIRT2/cPLA2α). Pulled-down beads were subjected to the HDAC assay in vitro by measuring the release of 3H-labeled acetyl groups from a histone H4 peptide (aa 1 to 21) under the conditions indicated. cpm, counts per minute. (F) Levels of Ac-α-tubulin were detected by Western blotting in total lysates of control HEK293 cells treated with control scrambled or cPLA2α siRNA. Intensities of Ac-α-tubulin bands were quantified by ImageJ (n = 3). (G) cPLA2α−/− MEFs were infected with Ad-LacZ or Ad-cPLA2α and treated with cycloheximide. Cell lysates were analyzed at the indicated time points by Western blotting for SIRT2 levels. ERK was used as a loading control. (H) SIRT2 mRNA levels were quantified in WT and cPLA2α−/− MEFs by quantitative reverse transcription-PCR. (I) cPLA2α-mediated AA release in the presence and absence of SIRT2 (upper panel). Cells were left untreated or were treated with ionophore A23187. Western blotting confirmed expression of proteins in transfected cells (lower panel). *, P < 0.05; **, P < 0.01. AU, arbitrary units.