Anti-NFATC4 抗体 (ab3447)

Immunocytochemistry/Immunofluorescence analysis of NFATC4 in A431 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3447 at a dilution of 1:100 overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFATC4 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of NFATC4 in HeLa cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3447 at a dilution of 1:20 overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFATC4 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of NFATC4 in U251 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control - right) or with ab3447 at a dilution of 1:20 overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFATC4 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Immunoprecipitation of NFATC4 was performed on MCF7 cells. The antigen:antibody complex was formed by incubating 500µg whole cell lysate with 3µg of rabbit polyclonal antibody recognizing NFATC4 (ab3447) overnight on a rocking platform at 4°C. The immune-complex was captured on 50µl Protein A/G Agarose. Captured immune-complexes were washed and proteins eluted with 5X Reducing Sample Loading Dye . Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1%Tween for at least 1 hour. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody at a dilution of 1:20000 for at least one hour. Membranes were washed and chemiluminescent detection performed.

Immunofluorescent analysis of NFATC4 using anti-NFATC4 polyclonal antibody ( ab3447) (shown in green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were then blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with a rabbit polyclonal antibody recognizing NFATC4 ( ab3447) at a dilution of 1:100 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.