Objective: SMAD-specific E3 ubiquitin-protein-ligase 2 (Smurf2) is a conserved gene across humans, mice, and zebrafish, whose protein is a ligase in the TGF-β signaling pathway. Due to the E3 ubiquitin ligase activity, it is known that Smurf2 has roles in multiple signaling pathways but there are no studies about changes in the protein levels of Smurf2 during brain aging. Previous studies showed that Smurf2 protein levels increase with aging in several tissues and we had found that Smurf2 gene expression increases in the old zebrafish brain. The first aim of this study is to investigate whether Smurf2 protein levels are altered in the aging brain. The second aim is to examine Smurf2 protein levels in a knockdown model. Methods: Zebrafish were maintained and raised in the standard conditions. Whole brain tissues were isolated from 6, 12, 18, 24, and 30 month-old, male and female zebrafish brains (AB/wildtype). For the second aim, Casper animals were used for cerebroventricular microinjections (CVMI) and whole brain tissues were isolated at 1, 4, 12 hours (hpi) and 1, 2, 3, 4 days post injection (dpi). Proteins from brain tissues were extracted and Western Blot analysis was used to determine differences in Smurf2 expression. Minimally three individual brain lysates in each age group and time-point were loaded in duplicates in every gel. An ANOVA analysis was applied (p<0.05). Results: Initial results demonstrated that Smurf2 protein levels are not altered with aging (p=0.313) nor gender (p=0.959). However, we expected to observe increased protein levels of Smurf2 in aged brains. Preliminary CVMI experiments demonstrated that the best knockdown efficiency is at 1 dpi (42% decreases) and changes in aging-related markers will be examined in ongoing experiments at 1 dpi. Conclusion: Taken together, these data suggest there may be post-transcriptional or even post-translational modifications in Smurf2 expression during aging. This work was supported by an EMBO Installation Grant. [ABSTRACT FROM AUTHOR]