AIP/ARA9 Antibody (35-2)

Western Blot: AIP/ARA9 Antibody (35-2) [NB100-127]

Western Blot: AIP/ARA9 Antibody (35-2) [NB100-127] - Effect of miR-34a (A) and anti-miR-34a (B) on endogenous AIP protein levels in HEK293 and in GH3 (C) cells 48 hours after transfection. Data are shown as mean +/- SEM, **, P<0.01 ***, P<0.001. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25658813/) licensed under a CC-BY license.

Flow Cytometry: AIP/ARA9 Antibody (35-2) [NB100-127]

Flow Cytometry: AIP/ARA9 Antibody (35-2) [NB100-127] - Intracellular flow cytometric staining of 1 x 10^6 CHO (A) and MCF-7 (B) cells using AIP/ARA9 antibody (dark blue). Isotype control shown in orange. An antibody concentration of 1 ug/1x10^6 cells was used.

Immunohistochemistry-Paraffin: AIP/ARA9 Antibody (35-2) [NB100-127]

Immunohistochemistry-Paraffin: AIP/ARA9 Antibody (35-2) [NB100-127] - AIP/ARA9 was detected in immersion fixed paraffin-embedded sections of human spleen using Mouse Anti-Human AIP/ARA9 (35-2) Monoclonal Antibody (Catalog # NB100-127) at 1:300 for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the cytoplasm in splenocytes.

Western Blot: AIP/ARA9 Antibody (35-2) [NB100-127] -

EPS15 is targeted via a SPOP/SPOPL binding consensus motif.(A) Cartoon of human EPS15 domain-organization and the amino-acid sequence. Indicated by color code are the SPOP/SPOPL binding site (red) and the lysine residue (yellow), which is ubiquitinated in a CRL3SPOPL–dependent manner in vivo. In addition, the amino-terminal Ca2+-binding EF-hand motifs (EH), the coiled-coil domain involved in dimerization and the two carboxy-terminal ubiquitin-interacting motifs (UIMs) involved in ubiquitin-binding are indicated. (B) EPS15 ubiquitin-profiling. Peptides containing EPS15 modification sites were quantified with LC-MS/MS after enrichment of the K-epsilon -GG motif from whole cell digests of HeLa cells treated with siSPOPL or siControl. Normalized precursor mass intensity profiles for EPS15 sites corresponding to K793, K801 and K693 are shown (raw data in Figure 4—figure supplement 1B). Quantification of the beta-Actin K113 and the polyubiquitin K11 linkage peptide control for comparable enrichment. Data are mean ± SD, N = 3. **p≤0.01. (C) Purified SPOPL was incubated as indicated with GST-tagged wild-type EPS15 or GST-EPS15 mutants, where the predicted SPOPL binding motifs have been mutated individually (GST-EPS15S605-S607A and EPS15S744-S746A, respectively), pulled down with glutathione sepharose (IP [GST]) and bound proteins were analyzed by Coomassie blue staining (upper panel) and immunoblotting (lower panels). Note that SPOPL readily binds to GST-EPS15 and GST-EPS15S605-S607A, but this interaction is strongly reduced with the GST-EPS15S744-S746A mutant. (D) HeLa cells stably expressing GFP-tagged wild-type EPS15, the EPS15S744-S746A or the EPS15K793R mutants from a doxycycline-inducible promoter were transfected as indicated (+) with control siRNA or siRNA depleting SPOPL. The levels of EPS15-GFP, EGFR and for control tubulin (TUB) were analyzed by immunoblotting with specific antibodies. Experiments were quantified in Fiji and the EPS15 levels plotted as fold-increase compared to controls. Data are mean ± SEM, N = 4. *p≤0.05. Note that SPOPL depletion does not further increase the levels of both EPS15 mutants. (E) Total cell extracts were prepared from HeLa cells expressing either GFP-tagged wild-type, the EPS15S744-S746A mutant or the EPS15K793R mutant in the presence (+) or absence (-) of HA-tagged SPOPL overexpression. The levels of EPS15-GFP, SPOPL-HA and control GAPDH were analyzed by immunoblotting. Note that overexpression of SPOPL-HA is able to induce degradation of wild-type but not the EPS15S744-S746A-GFP or the EPS15K793R-GFP mutant.DOI:https://dx.doi.org/10.7554/eLife.13841.009EPS15 is targeted via a SPOP/SPOPL binding consensus motif.(A) Alignments of the carboxy-terminal domains of EPS15 proteins from various species. Conserved SPOPL-binding motifs and putative ubiquitination sites are highlighted by yellow boxes. (B) Peptides containing EPS15 modification sites were quantified with LC-MS/MS after enrichment of the K-epsilon -GG motif from whole cell HeLa digests treated with siSPOPL and siControl. Raw intensities for each of the triplicate LC-MS/MS runs are shown with each of the siControl conditions scaled to 100% intensity. Normalized precursor mass intensity profiles for EPS15 sites corresponding to K793, K801 and K693 are shown, with only K793 showing significant downregulation in the depletion condition. Quantification of a peptide corresponding to beta-Actin K113 and the poly-ubiquitin K11 linkage peptide is also shown to demonstrate that enrichment variations did not influence the quantification of the EPS15 sites. Additionally, the total ion chromatographic intensities for each run are plotted to provide insight into the consistency of each of the separate experiments performed on different days. Data are mean ± SD, N = 3. (C) HeLa cell lines stably expressing wild-type EPS15-GFP, the EPS15S744-746A-GFP mutant or the EPS15K793R-GFP mutant from the inducible doxycycline-promoter were treated with doxycycline for 3 days, and analyzed by live cell imaging. Displayed are maximal projections of Z-stack acquisitions, fully covering cell height. Scale bar = 10 μm.DOI:https://dx.doi.org/10.7554/eLife.13841.010 Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/13841), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] -

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] - Aryl hydrocarbon receptor interacting protein (AIP) immunostaining.A & B—Examples of low AIP expression; C & D: Examples of high AIP expression; E—Normal human pituitary staining with omitting primary antibody (negative control); F—Normal human pituitary staining with AIP (positive control); Scale bar = 1000 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117107), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] -

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] - Aryl hydrocarbon receptor interacting protein (AIP) immunostaining.A & B—Examples of low AIP expression; C & D: Examples of high AIP expression; E—Normal human pituitary staining with omitting primary antibody (negative control); F—Normal human pituitary staining with AIP (positive control); Scale bar = 1000 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117107), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] -

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] - Aryl hydrocarbon receptor interacting protein (AIP) immunostaining.A & B—Examples of low AIP expression; C & D: Examples of high AIP expression; E—Normal human pituitary staining with omitting primary antibody (negative control); F—Normal human pituitary staining with AIP (positive control); Scale bar = 1000 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117107), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] -

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] - Aryl hydrocarbon receptor interacting protein (AIP) immunostaining.A & B—Examples of low AIP expression; C & D: Examples of high AIP expression; E—Normal human pituitary staining with omitting primary antibody (negative control); F—Normal human pituitary staining with AIP (positive control); Scale bar = 1000 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117107), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] -

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] - Aryl hydrocarbon receptor interacting protein (AIP) immunostaining.A & B—Examples of low AIP expression; C & D: Examples of high AIP expression; E—Normal human pituitary staining with omitting primary antibody (negative control); F—Normal human pituitary staining with AIP (positive control); Scale bar = 1000 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117107), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] -

Immunohistochemistry: AIP/ARA9 Antibody (35-2) [NB100-127] - Aryl hydrocarbon receptor interacting protein (AIP) immunostaining.A & B—Examples of low AIP expression; C & D: Examples of high AIP expression; E—Normal human pituitary staining with omitting primary antibody (negative control); F—Normal human pituitary staining with AIP (positive control); Scale bar = 1000 μm. Image collected & cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117107), licensed under a CC-BY license. Not internally tested by Novus Biologicals.