Afuco™ Anti-Human NCSTN ADCC Recombinant Antibody (A5226A), ADCC Enhanced (CAT#: AFC-714CL)

Figure 1 Characterization of anti NCT mAbs.

(a) Scatchard plot of A5201A and A5226A. From this plot, Kd value of A5201A and A5226A was calculated as 0.045 and 0.32 nM, respectively. (b) Immunoblot analysis of cell lysates using anti NCT mAbs. Mature and immature forms of NCT are indicated by white and black arrowheads, respectively. Note that only human NCT polypeptides were recognized by A5201A and A5226A. (c, d) Reactivity of mAbs against deglycosylated NCT. Immunoreactivities of both A5201A and A5226A were significantly reduced against endoglycosidase H resistant and sensitive species of NCT, which are indicated by white and black circles, respectively, in NKO cell lysates incubated with endoglycosidases (c). However, both antibodies recognized either the deglycosylated NCT species that was immunoisolated using anti NCT antibody (d).

Hayashi, I., Takatori, S., Urano, Y., Miyake, Y., Takagi, J., Sakata-Yanagimoto, M.,... & Wong, P. C. (2012). Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene,31(6), 787.

Figure 2 Immunoprecipitation analyses using anti NCT mAbs.

(a, b) Co immunoprecipitation of human NCT and the γ secretase components by anti NCT mAbs (indicated above the lanes) from CHAPSO solubilized HeLa (a), NKO, NKO expressing human NCT and wild type MEF (b) cell lysates. Immunoblot analyses were performed by antibodies shown below the lanes. Note that A5201A precipitated only immature NCT and APH 1aL, while A5226A captured all components of the γ secretase complex. (c) Schematic depiction of deletion mutants of NCT (Shirotani et al., 2003). (d) Reactivity of antibodies (shown at the right of the panels) against deletion mutant NCT in immunoprecipitation under the CHAPSO solubilized condition. All immunoblot analyses were performed by anti C terminus of NCT antibody N1660. (e) Amino acid sequence of human and mouse NCT around the region containing A5226A epitope. Species specific residues are shown in black background. (f) Reactivity of A5226A toward NCT ectodomain fragments with single residue substitution. Bindings of biotinylated A5226A toward various hGH NCT fusion proteins indicated are determined using a sandwich Enzyme linked immunosorbent assay format, normalized by the expression level deduced from a hGH immunoassay, and expressed as the relative value of the wild type human NCT.

Hayashi, I., Takatori, S., Urano, Y., Miyake, Y., Takagi, J., Sakata-Yanagimoto, M.,... & Wong, P. C. (2012). Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene,31(6), 787.

Figure 3 Immunocytochemical analysis using anti NCT mAbs.

(a) Immunocytochemical analysis of non permeabilized HeLa cells using anti NCT mAbs (green) and CTB (red). These images represent maximum projections of confocal optical sections. (b) Immunocytochemical analysis of permeabilized HeLa cells using anti NCT mAbs (gray scale in left columns and green in merged images) with antibodies against organelle markers (protein disulfide isomerase for ER; TGN46 for TGN) or CTB (red). Note that staining by A5201A and A5226A were well co localized with that by anti protein disulfide isomerase and CTB, respectively. (c) Specificity of anti NCT mAbs. Immunocytochemical analysis of NKO (left column) and NKO/hNCT cells (right column) revealed that neither A5201A nor A5226A showed any specific staining in NKO cells. (d) Co immunostaining of permeabilized HeLa cells using A5226A with anti PS1 antibody. (e) Immunocytochemical analysis of DAPT treated HeLa cells expressing NDE using A5226A (green) and anti NOTCH1 NEXT (Val1711) antibody that specifically recognizes S2 cleavage site (red). The higher magnification image of the boxed region is shown in the inset. Nuclei were conter labeled with DRAQ5 (blue) in all figures. Scale bars: 10 μm.

Hayashi, I., Takatori, S., Urano, Y., Miyake, Y., Takagi, J., Sakata-Yanagimoto, M.,... & Wong, P. C. (2012). Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene,31(6), 787.

Figure 4 Effect of A5226A treatment on living cells.

(a) Effects of anti NCT mAbs in the cell based γ secretase assay. Firefly luciferase activity was measured in HEK293 cells stably expressing amyloid precursor protein (open column) or Notch based substrate (filled column) fused with GAL4. Luciferase activity was normalized by fluorescence unit of co expressed EGFP, which is corresponding to cell viability. No change in fluorescence (that is cell viability) was observed by either mAb. A total of 100 nM DBZ was used as a positive control (n 3 6, **P<0.01). (b) Immunoblot analysis of HeLa cells treated with 100 μg/ml of anti NCT mAbs. (c) Effects of A5226A on the endocytosis of the γ secretase and amyloid precursor protein. HeLa cells were surface labeled with a disulfide cleavable biotinylation reagent, and then incubated for indicated times. Remaining reagents on the cell surface were stripped with a membrane impermeant reducing reagent, and the amount of the internalized proteins was analyzed by immunoblotting.

Hayashi, I., Takatori, S., Urano, Y., Miyake, Y., Takagi, J., Sakata-Yanagimoto, M.,... & Wong, P. C. (2012). Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene,31(6), 787.

Figure 5 Inhibitory effect of A5226A on γ secretase activity.

(a) Effects of anti NCT mAbs in an in vitro γ secretase assay. Solubilized HeLa cell membranes were co incubated with recom binant substrate, C100 FmH (Takahashi et al., 2003), in the presence of mAbs at indicated concentration. De novo generation levels of amyloid β peptide were shown (n 3, mean±s.e.m., *P<0.05). (b) Effects of anti NCT mAbs in a binding assay using recombinant NCT and substrate. Co incubated samples containing N100 FLAG/His and NCT V5/His with mAbs were immuno precipitated by antibodies indicated at the left of the panels and analyzed by immunoblot using antibodies below the panels.

Hayashi, I., Takatori, S., Urano, Y., Miyake, Y., Takagi, J., Sakata-Yanagimoto, M.,... & Wong, P. C. (2012). Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene,31(6), 787.

Figure 6 Anti cancer activity of A5226A.

(a) Effect of A5226A on the proliferation of A549 cells. Note that γ secretase activity dependent cell proliferation of A549 cells was specifically inhibited by A5226A (n 4, mean± s.e.m., *P<0.05, **P<0.01). (b) Effect of combination treatment of A5226A (100 μg/ml) and DAPT (100 μM) on the proliferation of A549 cells (n 3, mean±s.e.m., **P<0.01). (c) Effect of A5226A on the proliferation of DND 41 cells (n 3, mean± s.e.m., **P<0.01). (d) Immunoblot analysis of A5226A treated DND 41 cells treated with γ secretase inhibitors (10 μM DAPT and 100 nM DBZ) or mAbs (10 or 100 μg/mL) for 7 days. (e) Anti cancer effects of A5226A on xenograft models of DND 41 cells with concurrent injection of mAbs at a dose of 50 mg/kg/day.

Hayashi, I., Takatori, S., Urano, Y., Miyake, Y., Takagi, J., Sakata-Yanagimoto, M.,... & Wong, P. C. (2012). Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Oncogene,31(6), 787.

Figure 7 At the times indicated on the left cells were fixed and stained with anti-EEA1 (magenta) as Fig. 5. Colocalization of the γ-secretase at EEA1-positive compartment are depicted by asterisks. Bar, 10 μm.

(a) HT1080 cells treated with non-target or CALM siRNA duplexes were fixed and stained with the late endosomal marker anti-LAMP1 (magenta) and anti-Nct mAb A5226A for detection of endogenous γ-secretase (green). Bar, 10 μm. (b) Uptake assay using Anti-Nct mAb A5226A and Alexa647-conjugated transferrin (cyan) in HT1080 cells treated with non-target or CALM siRNA duplexes. At the times indicated on the left, cells were fixed and stained with anti-EEA1 (magenta). A5226A bound to endogenous γ-secretase (green) was visualized by an Alexa488-conjugated anti-mouse IgG secondary antibody. Colocalization of the γ-secretase at EEA1-positive compartment as well as the cell surface accumulation of the γ-secretase is depicted by asterisks and arrowheads, respectively. Bar, 10 μm. (c) After 480 min incubation in the uptake assay, HT1080 cells were fixed and stained with anti-Nct mAb A5226A (green) and anti-EEA1 (magenta). Majority of the γ-secretase were localized at non-EEA1-positive, lysosomal compartment. Bar, 10 μm. (d) Uptake assay using Anti-Nct mAb A5226A (green) in DMSO or Pitstop 2 (30 μM)-treated HeLa cells. At the times indicated on the left, cells were fixed and stained with anti-EEA1 (magenta). Colocalization of the γ-secretase at EEA1-positive compartment as well as the cell surface accumulation of the γ-secretase is depicted by asterisks and arrowheads, respectively. Bar, 10 μm. (e) HeLa cells treated with Pitstop 2 (30 μM) for 3 h were fixed and stained with anti-Nct mAb A5226A (green) and anti-CALM (magenta). Confocal image of cell surface accumulation of the γ-secretase was taken by Leica SP5 confocal microscope. Bar, 10 μm.

Kanatsu, K., Morohashi, Y., Suzuki, M., Kuroda, H., Watanabe, T., Tomita, T., & Iwatsubo, T. (2014). Decreased CALM expression reduces Aβ42 to total Aβ ratio through clathrin-mediated endocytosis of γ-secretase. Nature communications, 5, 3386.

Figure 8 Uptake assay using Anti-Nct mAb A5226A in HT1080 cells treated with non-target, CALM or APP siRNA.

At the times indicated on the left cells were fixed and stained with anti-EEA1 (magenta). Colocalization of the γ-secretase at EEA1-positive compartment are depicted by asterisks. Bar, 10 μm.

Kanatsu, K., Morohashi, Y., Suzuki, M., Kuroda, H., Watanabe, T., Tomita, T., & Iwatsubo, T. (2014). Decreased CALM expression reduces Aβ42 to total Aβ ratio through clathrin-mediated endocytosis of γ-secretase. Nature communications, 5, 3386.