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International Journal of Alzheimer’s Disease
Volume 2011, Article ID 729382, 11 pages
Research Article

TrkB Isoforms Differentially Affect AICD Production through Their Intracellular Functional Domains

Department of Biology, Drexel University, Philadelphia, PA 19027, USA

Received 2 September 2010; Revised 12 November 2010; Accepted 16 November 2010

Academic Editor: E. A. Rogaeva

Copyright © 2011 Sara Ansaloni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Figure 1: Effective shRNA mediated knock-down of TrkB in SH-SY5Y APP-Gal4 cells. The picture represent a Western blot analysis of whole cell lysates that have been probed for TrkB (all isoforms) with a pan-TrkB antibody (Santa Cruz Biotechnology) and an anti actin antibody (Sigma) as a loading control. The two bands observed in the upper panel represent full length TrkB (upper band) and truncated TrkB (lower band) respectively. Effective TrkB knock-down is achieved with the three constructs, NTRK2.1-3 targeting TrkB while a control (first lane) or a shRNA targeting APP (second lane) do not decrease TrkB levels. Due to differences in shRNA constructs the extent of knock-down is different (lane 4 versus 3 and 5). The construct referred to as shNTRK2.4 is not represented in the blots. This construct targets all TrkB isoforms, except the TrkB truncated (TrkB T) isoform, and when transfected, consistently caused cell death.

Figure 2: APP levels are decreased by knock-down of NTRK2 but not by knock-down of NTRK3. The picture represent a Western blot analysis of whole cell lysates that have been probed for APP (Sigma) and an anti actin antibody (Sigma) as a loading control. The upper panel represents levels of APP and the two bands correspond to N-O glycosylated (upper band) and to N-glycosylated (lower band) APP. The lower panel shows the actin levels that were used as loading control. APP levels are decreased by shRNA constructs targeting APP or NTRK2 (constructs NTRK2.1-3) but not by shRNA targeting NTRK3. NTRK3 encodes for TrkC a tyrosine receptor kinase that belongs to the same family as TrkB. This shows that the APP effect that we observe is specific to TrkB and cannot be recapitulated by TrkC even if the two proteins are related.

Figure 3: TrkB isoforms over-expressed as GFP fusions in SH-SY5Y APP Gal4 cells. Representative pictures of SH-SY5Y APP-Gal4 cells expressing the TrkB FL-GFP and TrkB T-GFP constructs as well as a farnesylated-GFP control plasmid. All the fluorescent cells were effectively transfected with the over-expression constructs. (A) Cells transfected with TrkB truncated full-length. (B) Cells transfected with TrkB truncated. (C) Cells transfected with a control GFP farnesylated construct. Assessment of transfection efficiency was performed via fluorescence microscopy before the cell lysates were collected for luciferase assay or Western blot analysis.

Figure 4: L-685 treatment of SH-SY5Y APP Gal4 cells causes accumulation of C83 Gal4. AICD Gal4 and C83 Gal4 levels in whole cell lysates were detected with anti APP antibody (A8717, Sigma) and identified thorugh molecular weight. AICD Gal4 is detectable in the vehicle (DMSO) treated cells (first three lanes) but not in the L-685 treated lanes. The signal of C83 Gal4 is greatly increased by L-685 treatment compared to vehicle (DMSO) treated cells.

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