Western blotting analysis using Nicastrin antibody (Cat#61775). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with Nicastrin antibody (Cat#61775, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Western blotting analysis using Nicastrin antibody (Cat#61775). Nicastrin expression in wild type (WT) and Nicastrin shRNA knockdown (KD) HT-1080 cells with 30 μg of total cell lysates. β-Tubulin serves as a loading control. The blot was incubated with Nicastrin antibody (Cat#61775, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Immunocytochemical staining of HepG2 cells with Nicastrin antibody (Cat#61775, 1:1,000). Nuclei were stained blue with DAPI; Nicastrin was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Medium. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol. Scale bar: 20 μm.
Immunocytochemical staining of HT-1080 cells using Nicastrin antibody (Cat#61775, 1:1,000), Top panel: wild-type (WT); Bottom panal: Nicastrin shRNA knockdown (KD). Nuclei were stained blue with DAPI; Nicastrin was stained magenta with Alexa Fluor® 647. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol. Scale bar, 20 μm.
Flow cytometric analysis of Nicastrin expression in HepG2 cells using Nicastrin antibody (Cat#61775, 1:2,000). Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use. Green, isotype control; red, Nicastrin.
Validation of Nicastrin knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HT1080 cells were stained with Nicastrin antibody (Cat#61775, 1:2,000) and analyzed using BD flow cytometer. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use.
