NFAT5 Antibody

Western Blot: NFAT5 Antibody [NB120-3446]

Western Blot: NFAT5 Antibody [NB120-3446] - Analysis of NFAT5 was performed by loading 25ug of various whole cell lysates 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 probed with a rabbit polyclonal antibody recognizing NFAT5 at a dilution of 1:1000 overnight at 4C on a rocking platform. 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 using Super Signal West Dura.

Western Blot: NFAT5 Antibody [NB120-3446]

Western Blot: NFAT5 Antibody [NB120-3446] - Analysis of human NFAT5 from transfected BHK cell lysate.

Western Blot: NFAT5 Antibody [NB120-3446]

Western Blot: NFAT5 Antibody [NB120-3446] - Western blot image and corresponding densitometry analyses showing TonEBP/NFAT5 levels under different osmotic conditions in wild-type and Kif3a null MEFs. Kif3a null MEFs show slightly attenuated hyperosmotic increase but unaffected hypoosmotic decrease in TonEBP expression. Image collected and cropped by CiteAb from the following publication (nature.com/articles/s41598-019-51939-7), licensed under a CC-BY license.

Immunocytochemistry/ Immunofluorescence: NFAT5 Antibody [NB120-3446]

Immunocytochemistry/Immunofluorescence: NFAT5 Antibody [NB120-3446] - Analysis of NFAT5 in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a NFAT5 polyclonal antibody at a dilution of 1:20 overnight at 4C, 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.

Immunocytochemistry/ Immunofluorescence: NFAT5 Antibody [NB120-3446]

Immunocytochemistry/Immunofluorescence: NFAT5 Antibody [NB120-3446] - Analysis of NFAT5 in MCF-7 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a NFAT5 polyclonal antibody at a dilution of 1:200 overnight at 4C, 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.

Immunocytochemistry/ Immunofluorescence: NFAT5 Antibody [NB120-3446]

Immunocytochemistry/Immunofluorescence: NFAT5 Antibody [NB120-3446] - Analysis of NFAT5 in NIH-3T3 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a NFAT5 polyclonal antibody at a dilution of 1:20 overnight at 4C, 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.

Immunohistochemistry-Paraffin: NFAT5 Antibody [NB120-3446]

Immunohistochemistry-Paraffin: NFAT5 Antibody [NB120-3446] - Normal biopsies of deparaffinized human brain tissue.

Immunoprecipitation: NFAT5 Antibody [NB120-3446]

Immunoprecipitation: NFAT5 Antibody [NB120-3446] - Analysis of NFAT5 was performed on U2OS cells. The antigen:antibody complex was formed by incubating 500ug whole cell lysate with 3ug of rabbit polyclonal antibody recognizing NFAT5 overnight on a rocking platform at 4C. The immune-complex was captured on 50ul 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 using Super Signal West Dura.

Western Blot: NFAT5 Antibody [NB120-3446] -

Western Blot: NFAT5 Antibody [NB120-3446] - Knockdown of Ift88 or Kif3a in NP cells does not affect hyperosmotic upregulation of TonEBP. (a) TonEBP/Nfat5 mRNA levels in NP cells with Ift88 knockdown (n ≥ 5). (b) Western blot image showing increased TonEBP expression in response to hyperosmolarity (550 mOsm/kg H2O) independently of Ift88 knockdown. (c) Densitometry analyses of TonEBP with Ift88 knockdown (n ≥ 4). (d) TonEBP/Nfat5 mRNA levels in NP cells with Kif3a knockdown (n ≥ 3). (e) Western blot image showing that hyperosmotic induction of TonEBP is maintained after Kif3a knockdown. (f) Densitometry analyses of TonEBP after Kif3a knockdown (n ≥ 4). Data are represented as scatter plots (mean ± SEM). ns = not significant. One-way ANOVA or Kruskal-Wallis test with Sidak’s or Dunn’s multiple comparison test was used based on the distribution of the data to determine statistical significance. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1-1 for un-cropped Western blot images. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31664118), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: NFAT5 Antibody [NB120-3446] -

Western Blot: NFAT5 Antibody [NB120-3446] - NP cells do not induce autophagy in response to hyperosmotic stimulus in an ex vivo disc organ culture model. (a) A schematic depicting ex vivo rat intervertebral disc organ culture model. (b) H&E & alcian blue staining of discs cultured under iso- (330 mOsm/kg H2O) or hyperosmotic (500 mOsm/kg H2O) conditions showing that NP maintained its structure & cellular morphology. Scale bar: 100 μm. (c) Western blot analysis of tissue proteins from NP or AF (annulus fibrosus) compartments of the organ culture discs. The level of TonEBP increased with hyperosmotic stimulus only in the NP. However, the levels of LC3-II, SQSTM1, ATG12-ATG5, BECN1, as well as pULK1 Ser757 did not change with hyperosmolarity in both NP & AF. (d–f) Densitometric analyses of multiple Western blots represented in (c). Bars represent mean ± SEM (n = 3; For each independent experiment, one motion segment per group was used for histology & 6 motion segments per group were used for tissue protein Western blot). Student t test was used to determine statistical significance. NS, non-significant. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28674405), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: NFAT5 Antibody [NB120-3446] -

Western Blot: NFAT5 Antibody [NB120-3446] - TonEBP does not control autophagy in NP cells. (a) Western blot analysis of NP cells transduced with a lentivirus expressing either control shRNA or shTonEBP plasmid showed that TonEBP silencing did not affect the levels of LC3-II, ATG12-ATG5, & BECN1. The levels of SQSTM1 increased with TonEBP silencing. (b–f) Densitometric analyses of multiple Western blots shown in (a). (g) Western blot analysis of ULK1 activation status showed that the levels of pULK1 Ser757 was slightly lower, while that of pULK1 Ser777 remained unaffected after TonEBP knockdown under hyperosmotic conditions. (h,i) Densitometric analyses of multiple western blots shown in (g). Bars represent mean ± SEM (n = 4). Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. NS, non-significant. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28674405), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: NFAT5 Antibody [NB120-3446] -

Western Blot: NFAT5 Antibody [NB120-3446] - Hyperosmolarity does not upregulate the levels of canonical autophagic markers. (a) Western blot analysis of NP cells cultured under increasing osmolarity (330–600 mOsm/kg H2O) showed that the levels of LC3-II, SQSTM1, ATG12-ATG5, & BECN1 did not change by hyperosmolarity. However, TonEBP expression increased under hyperosmotic condition. (b–d) Densitometric analyses of multiple Western blots represented by (a) confirmed significant induction of TonEBP, while LC3-II & SQSTM1 levels remained unaltered (n = 5). (e) Western blot analysis of NP cells cultured under hyperosmotic condition for increasing lengths of time demonstrated that LC3-II, SQSTM1, ATG12-ATG5, & BECN1 levels were unaffected by hyperosmolarity up till 72 h. (f–i) Densitometric analyses of multiple Western blots shown in (e) (n = 3). Bars represent mean ± SEM. One-way ANOVA with Sidak’s multiple comparisons test was used to determine statistical significance. NS, non-significant. Western blot images were cropped & acquired under same experimental conditions. See Supplementary Fig. S1 for examples of uncropped images. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28674405), licensed under a CC-BY license. Not internally tested by Novus Biologicals.