antibody against itch  (Proteintech)

Journal: Oncotarget

Article Title: Interferon regulatory factor 4 attenuates Notch signaling to suppress the development of chronic lymphocytic leukemia

doi: 10.18632/oncotarget.9596

Figure Lengend Snippet: A. Western-blot analysis to detect the levels of Hes1 protein in IRF4 −/− Vh11 CLL cells compared to IRF4 +/+ Vh11 B cells isolated from spleen. Each lane represents CLL cells from an IRF4 −/− Vh11 mouse. B. Western-blot to detect the levels of Notch2 and Notch1 proteins in IRF4 −/− Vh11 CLL cells. Each lane represents an individual CLL sample. Thymus is used as a positive control for Notch1 protein and actin is used as loading control. C. Histograms showing Notch2 cell surface staining in IRF4 −/− Vh11 CLL cells compared to IRF4 +/+ Vh11 B cells as detected by Flow cytometry. Left panel shows isotype control antibody (IgG) staining and right panel shows Notch2 staining. Gray line represents gating on IRF4 +/+ Vh11 B cells and black line indicates IRF4 −/− Vh11 CLL cells. The data shown is representative of at least three independent experiments.

Article Snippet: The antibodies against Notch1, Notch2, Itch, Nedd4 and Hes1 were purchased from Cell signaling Technologies.

Techniques: Western Blot, Isolation, Positive Control, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Interferon regulatory factor 4 attenuates Notch signaling to suppress the development of chronic lymphocytic leukemia

doi: 10.18632/oncotarget.9596

Figure Lengend Snippet: A. Histograms showing Notch2 staining in CLL cells isolated from blood and spleen of NSG mice fed with (black line) or without dox water for 3 weeks (gray line). B. Western-blot analysis to detect Notch and IRF4 levels in CLL cells isolated from NSG mice fed with or without dox water for 3 weeks. The numbers below represents normalized relative expression. B cells isolated from B6 mice are used as a measure of endogenous levels of IRF4. Actin is used as loading control. C. Bar graph representing the relative mRNA expression of Hes1, Nedd4 and Fbxw7 in CLL cells isolated from NSG mice fed with or without dox water for 3 weeks. D. Western-blot analysis to measure Nedd4 and Itch protein levels in NSG mice fed with or without dox. E. Bar graph showing the relative mRNA expression of Nedd4 in four different IRF4 −/− Vh11 CLL samples compared to B cells isolated from wildtype B6 and IRF4 +/+ Vh11 mice. F. Western-blot analysis to measure the levels of Nedd4 protein in IRF4 −/− Vh11 CLL samples compared to IRF4 +/+ Vh11 B cells. The numbers at the bottom represents Nedd4 expression measured by densitometric analysis using ImageJ software. Actin is used as the loading control. * p value ≤0.01 ** p value ≤0.05.

Article Snippet: The antibodies against Notch1, Notch2, Itch, Nedd4 and Hes1 were purchased from Cell signaling Technologies.

Techniques: Staining, Isolation, Western Blot, Expressing, Software

Journal: Oncotarget

Article Title: Interferon regulatory factor 4 attenuates Notch signaling to suppress the development of chronic lymphocytic leukemia

doi: 10.18632/oncotarget.9596

Figure Lengend Snippet: A. Western-blot showing the levels of Nedd4, Notch2, Hes1 and IRF4 in IRF4 −/− B1 cells compared to IRF4 +/+ B1 cells. The numbers below represent normalized relative expression calculated by densitometric quantification of respective proteins. B. Bar graph showing the relative mRNA expression of Nedd4 and Fbxw7 in IRF4 −/− and IRF4 +/+ B1 cells. C. Bar graph showing the relative mRNA expression of Nedd4 and Fbxw7 in IRF4 −/− and IRF4 +/+ B2 cells. D. Western-blot analysis to detect the levels of Pu.1 in B1 cells isolated from PC and B2 cells isolated from spleen of wild type mice. E. Flow cytometry analysis using intracellular staining to measure the levels of IRF4 in PC B1 cells and splenic B2 cells. The histograms represents intracellular staining with isotype control antibody (left panel) and with IRF4 antibody (right panel). Gray line represents B2 cells and Black line represents B1 cells. Cells were gated specifically on B1 and B2 populations based on IgM and B220 staining. * p value ≤0.01.

Article Snippet: The antibodies against Notch1, Notch2, Itch, Nedd4 and Hes1 were purchased from Cell signaling Technologies.

Techniques: Western Blot, Expressing, Isolation, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Interferon regulatory factor 4 attenuates Notch signaling to suppress the development of chronic lymphocytic leukemia

doi: 10.18632/oncotarget.9596

Figure Lengend Snippet: A. Western-blot showing Nedd4, IRF4, Notch2, Notch1, Hes1 and Actin expression upon IRF4 knockdown using an IRF4 specific (IRF4) or scrambled control (Con) siRNA in normal human B cells isolated from healthy donors. The numbers below represents normalized relative expression. B. Bar graph showing relative mRNA expression of IRF4, Nedd4, Hes1 and Fbxw7 in normal human B cells in control versus IRF4 specific siRNA. C. Western blot analysis showing Nedd4 and IRF4 expression in human CLL samples represented by each individual lane. D. Scatter plot to show the correlation between IRF4 and Nedd4 protein expression in human CLL cells. The dotted line represents the linear trend line. Pearson correlation coefficient (r) value is 0.865. E. Western-blot analysis of Nedd4, Itch, Notch1, Notch2 and IRF4 following Nedd4 knockdown using siRNA in human Mec-1 CLL cells. Knockdown with scrambled siRNA is used as controls (con). The numbers below represent the normalized relative expression of respective genes measured by densitometric analysis. (PBMCs).*p value ≤0.0001 ** p value ≤0.01.

Article Snippet: The antibodies against Notch1, Notch2, Itch, Nedd4 and Hes1 were purchased from Cell signaling Technologies.

Techniques: Western Blot, Expressing, Isolation

Journal: Oncotarget

Article Title: The anti-HER3 (ErbB3) therapeutic antibody 9F7-F11 induces HER3 ubiquitination and degradation in tumors through JNK1/2- dependent ITCH/AIP4 activation

doi: 10.18632/oncotarget.9455

Figure Lengend Snippet: ( A ) Protein sequence alignment of HER3 and HER4 C-terminal tails showing the homology between the HER3 972 PPRY 975 motif and the ITCH-binding motif 1053 PPAY 1056 in HER4. ( B ) Serum-starved BxPC3 cells were incubated with 50 μg/mL 9F7-F11 for 30 min or with 100ng/mL NRG-1β for 30 min or 1 hr. After immunoprecipitation (ip) of 2 mg of total protein extracts with an anti-HER3 monoclonal antibody against HER3 C-terminal tail (HER3 Ab), ITCH, HER3 and NEDD4 were detected by western blotting. ( C ) Serum-starved BxPC3 cells were pre-incubated with 10 μM MG132 for 4 hr before incubation with 50 μg/mL 9F7-F11 with or without 100 ng/mL NRG-1β for the indicated times. After immunoprecipitation with HER3 Ab, HER3 ubiquitination status was assessed by western blotting using a specific anti-ubiquitin antibody (Ub). ( D ) MDA-MB468 cells were transfected with the Myc-ITCH plasmid and the HA-Ubiquitin plasmid or not for 24 hr. Then, cells were incubated with 20 μM MG132 for 5 hr before addition of 50 μg/mL 9F7-F11 for 2 hr. After immunoprecipitation with HER3 Ab, HER3 was probed with an ubiquitin-specific antibody as in (C). WCL, whole cell lysate.

Article Snippet: The mouse monoclonal antibody against ITCH (ref. 611199) was from BD Biosciences (San José, CA).

Techniques: Sequencing, Binding Assay, Incubation, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: The anti-HER3 (ErbB3) therapeutic antibody 9F7-F11 induces HER3 ubiquitination and degradation in tumors through JNK1/2- dependent ITCH/AIP4 activation

doi: 10.18632/oncotarget.9455

Figure Lengend Snippet: Pancreatic BxPC3 ( A ) and prostatic DU145 ( B ) cancer cells were transfected with 10 nM Scramble Control siRNA (siSC) or the anti- ITCH/AIP4 siRNA (siITCH) for 72 hr, serum-starved and then incubated with 50 μg/mL 9F7-F11 or with 100 ng/mL NRG-1β for 4 hr. ITCH, HER3, AKT, ERK1/2, NEDD4 and Nrdp1 protein expression and ITCH, HER3, AKT and ERK1/2 phosphorylation were assessed in whole cell lysates (WCL) by western blotting. Band signal intensity (SI) was quantified with ImageJ, and β-tubulin was used as loading control. ( C ) BxPC3 cells were transfected with 10 nM siSC or siITCH for 72 hr, and then pre-incubated with 10 μM MG132 for 4 hr before addition of 9F7-F11 or NRG1-β for 4 hr. After immunoprecipitation with HER Ab, the HER3 ubiquitination status was analyzed by western blotting with a specific poly-ubiquitin chain antibody. HER3 and ITCH proteins were also detected by using specific antibodies.

Article Snippet: The mouse monoclonal antibody against ITCH (ref. 611199) was from BD Biosciences (San José, CA).

Techniques: Transfection, Incubation, Expressing, Western Blot, Immunoprecipitation