All lanes : Anti-NFAT5 antibody (ab3446) at 1/1000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 4 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 5 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 6 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
Lane 7 : HeLa (Human epithelial adenocarcinoma cell line) whole cell lysate
Lane 8 : COS-7 (African green monkey kidney fibroblast-like cell line) whole cell lysate
Lane 9 : EL4 (Mouse thymic lymphoma cell line) whole cell lysate
Lane 10 : C2C12 (Mouse myoblast cell line) whole cell lysate
Lane 11 : NRK (Rat kidney normal tissue) whole cell lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Goat anti-rabbit-HRP secondary antibody at 1/20000 dilution

Predicted band size: 160 kDa

Western blot analysis of NFAT5 was performed by loading samples onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were incubated with ab3446 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a secondary antibody for at least one hour. Membranes were washed and chemiluminescent detection performed.

Immunofluorescence analysis of NFAT5 in HeLa (Human epithelial adenocarcinoma cell line) cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3446 at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFAT5 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Immunohistochemistry was performed on normal biopsies of deparaffinized human skeletal muscle tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 duution with ab3446 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunoprecipitation of NFAT5 was performed on U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate (lane 2). The antigen:antibody complex was formed by incubating 500 µg whole cell lysate with 3 µg of ab3446 overnight on a rocking platform at 4°C. The immune-complex was captured on 50 µL Protein A/G Plus 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 1/20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed.

Lane 1: Only cell lysate.

Immunoprecipitation of NFAT5 was performed on U2OS cells. The antigen:antibody complex was formed by incubating 500µg whole cell lysate with 3µg of ab3446 overnight on a rocking platform at 4°C. The immune-complex was captured on 50µl Protein A/G Plus 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 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed.

Immunofluorescence analysis of NFAT5 in NIH/3T3 (Mouse embryo fibroblast cell line) cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3446 at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFAT5 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 NFAT5 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% Blocker BSA for 15 minutes at room temperature. Cells were probed with ab3446 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.

Immunohistochemistry was performed on normal biopsies of deparaffinized human brain tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with ab3446 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemical analysis of rat spinal tissue, staining NFAT5 with ab3446. Sections were incubated with primary antibody (1/100) overnight at 4°C before incubating with a biotinylated secondary antibody. Staining was detected using DAB.

Immunofluorescence analysis of NFAT5 in MCF7 (Human breast adenocarcinoma cell line) cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3446 at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFAT5 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.