JCS042226 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Table S1. Antibodies used in this study.

• Supplemental Figure S1 -

  Fig. S1. Intracellular Aβ rather than APP colocalizes with Omi in Tg2576 mouse brain tissue. (A) No colocalization between APP and Omi was detected in Tg2576 mouse brain tissue. APP was detected using primary mouse anti-APP and secondary Alexa-Fluor-488-conjugated goat anti-mouse IgG (green) antibodies. (B) Intracellular Aβ colocalized with Omi in Tg2576 mouse brain tissue. Both intracellular Aβ and APP were detected by primary mouse 6E10 and secondary Alexa-Fluor-488-conjugated goat anti-mouse IgG (green) antibodies. (C) Non-Tg littermate brain tissue was not staining with the 6E10 antibody, which is specific to human Aβ and APP. Omi was detected by primary rabbit anti-Omi and secondary Alexa-Fluor-568-conjugated goat anti-rabbit IgG (red) antibodies. Confocal images were obtained with a Carl Zeiss LSM510 Meta microscope. Scale bar: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. The PDZ domain is responsible for the interaction of Omi with oligomeric Aβ. (A) OmiS306A lost its proteolytic activity towards β-casein, whereas Omi degraded β-casein in a dose-dependent manner. β-casein (2 µM) was incubated with 0, 3.1, 6.3, 12.5, 25 or 50 nM Omi (lanes 1-6) and 100 nM OmiS306A (lane7), respectively, at 37°C for 2 hours. M, marker. (B) OmiS306A also preferentially bound to oligomeric Aβ. The same amounts of reverse Aβ (Aβ42-1), monomeric Aβ or oligomeric Aβ were spotted on the immunoblot membrane as indicated. The immunoblot membrane was hybridized with OmiS306A protein and then probed with an anti-Omi antibody. (C) OmiΔPDZ showed strong β-casein-hydrolysis activity. β-casein (2 µM, lane 1) was completely degraded by incubation with 10 or 20 nM OmiΔPDZ (lane 2, 3) at 37°C for 2 hours. M, marker. (D) OmiΔPDZ could bind neither monomeric nor oligomeric Aβ. The same amounts of reverse Aβ (Aβ42-1), monomeric Aβ or oligomeric Aβ were spotted on immunoblot membranes as indicated. The left immunoblot was initially hybridized with the OmiΔPDZ protein and then probed with an anti-Omi antibody. The middle and right immunoblots were directly detected with an anti-oligomer (A11) antibody specific to the oligomeric Aβ forms or an anti-Aβ (6E10) antibody that recognizes both monomeric and oligomeric Aβ.

• Supplemental Figure S3 -

  Fig. S3. Primary cortical neurons grow and form processes normally after transfection by Lipofectamine 2000. (A,B) Primary cortical neurons were transfected with 40 nM BLOCK-iT Fluorescent Oligo using Lipofectamine 2000 for 5 hours. Using the BLOCK-iT Fluorescent Oligo as an indicator of transfection, over 90% of the neurons were fluorescein-positive. (C,D) Primary cortical neurons transfected with 40 nM BLOCK-iT Fluorescent Oligo for 5 hours were then incubated for an additional 48 hours. (E) Primary cortical neurons transfected with 40 nM Omi siRNA for 5 hours were then incubated for an additional 48 hours. (F) Non-transfected primary cortical neurons were incubated under the same conditions as in C and E. All images were visualized by a Nikon ECLIPSE TE2000-S fluorescence microscope. Scale bar: 25 µm.