Fig 1: Effect of PLD or dioleoyl-PA on PARN deadenylation activity. (A,B) Validation study for in vitro PARN deadenylase activity. (A) Radiolabeled A15 RNA substrate was deadenylated by recombinant PARN with respect to the A15-only control. Deadenylation is evidenced by a greater mobility of radiolabeled spots and the appearance of smears versus the negative control of A15 alone. (B) Recombinant PARN, but not recombinant PAN2 is able to deadenylate A15. (C) Coomassie-stained gel indicating the high purity of the recombinant, purified proteins used. (D) PARN deadenylase activity as measured in lysates from COS-7 cells that were silenced with 150 ng of either control RNA (SiNeg), siPARN RNA or siPAN2 RNA. (E) PARN activity in overexpressing cells was concentration dependent from cell lysates in comparison with the deadelynase-inactive mutant PARN-H377A. (F) PARN activity of lysates prepared from COS-7 cells overexpressing PLD2 incubated with or without dioleolyl-PA. (G) PLD activity of COS-7 cells overexpressing PLD2 alone (control) or co-overexpressed with PARN-WT or the PARN mutant. Data are presented as means+s.e.m. The difference between means was assessed by single-factor ANOVA. *P<0.05, significant increase between samples and controls; #P<0.05, significant decrease between samples and controls.