Figure 4.
AIMP2 accumulation leads to PARP1 activation and cell death.
(a) Viability of SH-SY5Y cells transfected with AIMP2 and mock DNA. DPQ, 10 μM; ZVAD, 50 μM. n = 4 per group.
(b) Western blot analysis of PARP1 self-PARsylation and PAR conjugation to proteins in SH-SY5Y cells (Empty triangle, FLAG-AIMP2; filled triangle, endogenous AIMP2).
(c) Immunoprecipitation of FLAG-AIMP2 and endogenous PARP1 in SH-SY5Y cells.
(d) Anti-PARP1 pull-down of recombinant AIMP2 (rAIMP2) and immunopurified PARP1 (IP PARP1).
(e) Anti-FLAG pull-down of recombinant PARP1 (rPARP1) and recombinant AIMP2-FLAG (rAIMP2-FLAG).
(f) Subcellular localization of AIMP2 and PARP1 in SH-SY5Y cells.
(g) Confocal microscopic images in SH-SY5Y cells transfected with FLAG-AIMP2.
(h) Co-immunoprecipitation of AIMP2 using PARP1 antibody in the ventral midbrain (VM) of mice.
(i) Co-immunoprecipitation of PARP1 using FLAG antibody in the ventral midbrain of mice.
(j) In vitro PARP1 activation assay (n = 5 per group).
(k) Subcellular localization of AIMP2 and PARP1 in the ventral midbrain of mice.
(l) Western blots of AIMP2 and PAR in the ventral midbrain of 12 month old mice.
Quantified data (a, j) are expressed as mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, (a) ANOVA test followed by Student-Newman-Keuls post-hoc analysis, or (j) Mann-Whitney U test. Similar results were reproduced in multiple experiments (c, d, e, f, h, i, k). β-actin serves as a loading control (b, c, h, i, l). Full length blots are presented in Supplementary Figure 14 (b–f, h–i, k–l).