Date of download: 6/22/2016 The Association for Research in Vision and Ophthalmology Copyright © All rights reserved. From: Simultaneous Cell Death and Upregulation of Poly(ADP-Ribose) Polymerase-1 Expression in Early Postnatal Mouse Retina Invest. Ophthalmol. Vis. Sci ;52(10): doi: /iovs From: Simultaneous Cell Death and Upregulation of Poly(ADP-Ribose) Polymerase-1 Expression in Early Postnatal Mouse Retina Invest. Ophthalmol. Vis. Sci ;52(10): doi: /iovs Figure Legend: PARP-1 expression during postnatal development of the retina. (A) Western blot analysis of PARP-1 (by using the monoclonal anti–PARP-1 antibody) representative of three independent experiments. High levels of PARP-1 protein (113 kDa) are present at P3 and P7, decreasing thereafter. Bands at P28 and P60 are not seen because the immunoblot was exposed for a short time to avoid strong overexposure of bands at P3 and P7. Overexposed immunoblot showing the P60 PARP-1 band is seen in Supplementary Figure S1. The presence of PARP-1 fragments of 89 kDa at P3 to P7 indicates caspase activity (cleaving part of 113 kDa PARP-1). β-Tubulin was used as a loading control. (B) Densitometric analysis of complete PARP-1 form (113 kDa) in three different experiments. PARP-1 protein is increased in P3 and P7 retinal extracts, was strongly diminished in extracts older than P7, and was still present at P60. Densitometric values were expressed as relative levels with respect to the level at E18. *P < 0.01; significant differences between marked values. (C) Western blot analysis of PARP-1 expression (monoclonal anti–PARP-1 antibody) between P7 and P15 showing a gradual decrease in PARP-1 protein. The blot is representative of three independent experiments. (D) Western blot analysis of PARP-1 expression (using polyclonal anti–PARP-1 antibody) between P3 and P21 showed findings similar to those revealed with the monoclonal antibody. The blot is representative of three independent experiments. (E) Western blot analysis of PARP-1 expression in the retinas of P7 and P14 BALB/c mice. The blot is representative of three independent experiments and was obtained by using the polyclonal anti–PARP-1 antibody. Strong decrease in PARP-1 protein is seen in BALB/c mice between P7 and P14, similar to that observed in C57BL/6 mice. (F) Quantitative analysis of PARP-1 mRNA expression by real-time PCR using SYBR Green real-time analysis during retinal development. Histogram represents the CT (mean ± SEM) of three real-time PCR experiments conducted in triplicate; results were normalized to the expression of 18S rRNA. PARP-1 mRNA increases from P0 to P7, peaks at P7, and is subsequently downregulated. Note that the increase in PARP-1 mRNA coincides with increased PARP-1 protein levels. *P < 0.05 and **P < 0.0; significant differences between marked values. (G) Representative gel of three independent experiments on agarose gel electrophoresis of PARP-1 mRNA from P0, P7, P14, and P21 retinas. PARP-1 cDNA amplification products were run on 1.5% agarose gel, and the bands were digitalized. PARP-1 amplification products were referred to the corresponding 18S rRNA bands.