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Proteintech otub1 primary antibody

circ_0005185 binds to the 196–247 aa region of <t>OTUB1</t> and the 32–83 aa region of RAB8A. A) Silver staining image of an SDS‐PAGE gel displaying the isolation of circ_0005185/protein complexes from the RNA pull‐down experiment in DU145 cells; the red box highlights the specific protein bands enriched in the pull‐down complex by the circ_0005185 probe compared to the NC probe. B) RNA pull‐down assays coupled with western blot analysis confirmed the binding of circ_0005185 to OTUB1 and RAB8A proteins. C,D) RIP experiments utilizing OTUB1 and RAB8A primary antibodies or IgG were performed to assess the enrichment of circ_0005185 with proteins in DU145 and 22RV1 cells. Western blot was used to detect OTUB1 and RAB8A proteins immunoprecipitated by their respective antibodies or IgG. E–F) Interaction profiles of various regions of OTUB1 and RAB8A proteins with circ_0005185 were analyzed using the catRAPID database (http://www.tartaglialab.com/) to gain insights into their binding specificity. G) RIP assays were employed to quantitatively determine the enrichment of circ_0005185 within the 51–102, 101–152, 146–197, and 196–247 amino acid (aa) regions of the OTUB1 protein. H) Similarly, RIP assays were employed to quantitatively assess the enrichment of circ_0005185 within the 32–83, 51–102, 101–152, and 126–177 aa regions of the RAB8A protein. Data are presented as the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001, ns, not significant).

Otub1 Primary Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/otub1+primary+antibody/RAB8A+Antibody/pmc11848605-289-12-15
Average 93 stars, based on 44 article reviews

otub1 primary antibody - by Bioz Stars, 2026-09

93/100 stars

Images

1) Product Images from "Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway"

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway

Journal: Advanced Science

doi: 10.1002/advs.202411675

circ_0005185 binds to the 196–247 aa region of OTUB1 and the 32–83 aa region of RAB8A. A) Silver staining image of an SDS‐PAGE gel displaying the isolation of circ_0005185/protein complexes from the RNA pull‐down experiment in DU145 cells; the red box highlights the specific protein bands enriched in the pull‐down complex by the circ_0005185 probe compared to the NC probe. B) RNA pull‐down assays coupled with western blot analysis confirmed the binding of circ_0005185 to OTUB1 and RAB8A proteins. C,D) RIP experiments utilizing OTUB1 and RAB8A primary antibodies or IgG were performed to assess the enrichment of circ_0005185 with proteins in DU145 and 22RV1 cells. Western blot was used to detect OTUB1 and RAB8A proteins immunoprecipitated by their respective antibodies or IgG. E–F) Interaction profiles of various regions of OTUB1 and RAB8A proteins with circ_0005185 were analyzed using the catRAPID database (http://www.tartaglialab.com/) to gain insights into their binding specificity. G) RIP assays were employed to quantitatively determine the enrichment of circ_0005185 within the 51–102, 101–152, 146–197, and 196–247 amino acid (aa) regions of the OTUB1 protein. H) Similarly, RIP assays were employed to quantitatively assess the enrichment of circ_0005185 within the 32–83, 51–102, 101–152, and 126–177 aa regions of the RAB8A protein. Data are presented as the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001, ns, not significant).

Figure Legend Snippet: circ_0005185 binds to the 196–247 aa region of OTUB1 and the 32–83 aa region of RAB8A. A) Silver staining image of an SDS‐PAGE gel displaying the isolation of circ_0005185/protein complexes from the RNA pull‐down experiment in DU145 cells; the red box highlights the specific protein bands enriched in the pull‐down complex by the circ_0005185 probe compared to the NC probe. B) RNA pull‐down assays coupled with western blot analysis confirmed the binding of circ_0005185 to OTUB1 and RAB8A proteins. C,D) RIP experiments utilizing OTUB1 and RAB8A primary antibodies or IgG were performed to assess the enrichment of circ_0005185 with proteins in DU145 and 22RV1 cells. Western blot was used to detect OTUB1 and RAB8A proteins immunoprecipitated by their respective antibodies or IgG. E–F) Interaction profiles of various regions of OTUB1 and RAB8A proteins with circ_0005185 were analyzed using the catRAPID database (http://www.tartaglialab.com/) to gain insights into their binding specificity. G) RIP assays were employed to quantitatively determine the enrichment of circ_0005185 within the 51–102, 101–152, 146–197, and 196–247 amino acid (aa) regions of the OTUB1 protein. H) Similarly, RIP assays were employed to quantitatively assess the enrichment of circ_0005185 within the 32–83, 51–102, 101–152, and 126–177 aa regions of the RAB8A protein. Data are presented as the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001, ns, not significant).

Techniques Used: Silver Staining, SDS Page, Isolation, Western Blot, Binding Assay, Immunoprecipitation

circ_0005185 facilitates the deubiquitination of RAB8A by mediating the interaction between OTUB1 and RAB8A. A) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185. B) The Co‐IP experiment used OTUB1 antibody to verify the binding between RAB8A and OTUB1, which increased after overexpression of circ_0005185. C) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185, while knockdown of OTUB1 rescued the level of RAB8A. D) The results of immunofluorescence showed the localization and expression of RAB8A in the control group and circ_0005185 overexpression group. E,F) The ubiquitination level of RAB8A was detected in DU145 and 22RV1 cells using ubiquitination antibodies. Overexpression of circ_0005185 led to decreased ubiquitination of RAB8A, while knockdown of OTUB1 resulted in increased ubiquitination of RAB8A. G,H) The regulation of ubiquitination at the K48 site of RAB8A by OTUB1 was detected in DU145 and 22RV1 cells using antibodies specific to the K48 ubiquitination site. I) Ubiquitination at the K63 site of RAB8A was detected in DU145 cells using antibodies specific to the K63 ubiquitination site.

Figure Legend Snippet: circ_0005185 facilitates the deubiquitination of RAB8A by mediating the interaction between OTUB1 and RAB8A. A) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185. B) The Co‐IP experiment used OTUB1 antibody to verify the binding between RAB8A and OTUB1, which increased after overexpression of circ_0005185. C) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185, while knockdown of OTUB1 rescued the level of RAB8A. D) The results of immunofluorescence showed the localization and expression of RAB8A in the control group and circ_0005185 overexpression group. E,F) The ubiquitination level of RAB8A was detected in DU145 and 22RV1 cells using ubiquitination antibodies. Overexpression of circ_0005185 led to decreased ubiquitination of RAB8A, while knockdown of OTUB1 resulted in increased ubiquitination of RAB8A. G,H) The regulation of ubiquitination at the K48 site of RAB8A by OTUB1 was detected in DU145 and 22RV1 cells using antibodies specific to the K48 ubiquitination site. I) Ubiquitination at the K63 site of RAB8A was detected in DU145 cells using antibodies specific to the K63 ubiquitination site.

Techniques Used: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Knockdown, Immunofluorescence, Expressing, Control, Ubiquitin Proteomics

RNA Binding Assay:

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway
Article Snippet: Prostate cancer cells were treated with IP lysis buffer (Leagene Biotechnology, Beijing, China) and stored in lysis buffer at −80 °C overnight. .. Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17‐701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein‐bound RNA, and IgG primary antibody was used as a control. .. A circRNA cDNA library was constructed using a PrimeScript RT kit and gDNA Eraser (TaKaRa Biotech, Dalian, China). qRT–PCR was performed to detect the RNA level of circ_0 005185, and western blotting was performed to detect the OTUB1 and RAB8A protein levels.

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway.
Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense RNA-Binding Protein Immunoprecipitation (RIP) Assay: Prostate cancer cells were treated with IP lysis buffer (Leagene Biotechnology, Beijing, China) and stored in lysis buffer at −80 °C overnight. .. Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein-bound RNA, and IgG primary antibody was used as a control. .. A circRNA cDNA library was constructed using a PrimeScript RT kit and gDNA Eraser (TaKaRa Biotech, Dalian, China). qRT–PCR was performed to detect the RNA level of circ_0 005185, and western blotting was performed to detect the OTUB1 and RAB8A protein levels.

Immunoprecipitation:

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway.
Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense RNA-Binding Protein Immunoprecipitation (RIP) Assay: Prostate cancer cells were treated with IP lysis buffer (Leagene Biotechnology, Beijing, China) and stored in lysis buffer at −80 °C overnight. .. Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein-bound RNA, and IgG primary antibody was used as a control. .. A circRNA cDNA library was constructed using a PrimeScript RT kit and gDNA Eraser (TaKaRa Biotech, Dalian, China). qRT–PCR was performed to detect the RNA level of circ_0 005185, and western blotting was performed to detect the OTUB1 and RAB8A protein levels.

Control:

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway.
Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense RNA-Binding Protein Immunoprecipitation (RIP) Assay: Prostate cancer cells were treated with IP lysis buffer (Leagene Biotechnology, Beijing, China) and stored in lysis buffer at −80 °C overnight. .. Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein-bound RNA, and IgG primary antibody was used as a control. .. A circRNA cDNA library was constructed using a PrimeScript RT kit and gDNA Eraser (TaKaRa Biotech, Dalian, China). qRT–PCR was performed to detect the RNA level of circ_0 005185, and western blotting was performed to detect the OTUB1 and RAB8A protein levels.

Image Search Results

Journal: Advanced Science

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway

doi: 10.1002/advs.202411675

Figure Lengend Snippet: circ_0005185 binds to the 196–247 aa region of OTUB1 and the 32–83 aa region of RAB8A. A) Silver staining image of an SDS‐PAGE gel displaying the isolation of circ_0005185/protein complexes from the RNA pull‐down experiment in DU145 cells; the red box highlights the specific protein bands enriched in the pull‐down complex by the circ_0005185 probe compared to the NC probe. B) RNA pull‐down assays coupled with western blot analysis confirmed the binding of circ_0005185 to OTUB1 and RAB8A proteins. C,D) RIP experiments utilizing OTUB1 and RAB8A primary antibodies or IgG were performed to assess the enrichment of circ_0005185 with proteins in DU145 and 22RV1 cells. Western blot was used to detect OTUB1 and RAB8A proteins immunoprecipitated by their respective antibodies or IgG. E–F) Interaction profiles of various regions of OTUB1 and RAB8A proteins with circ_0005185 were analyzed using the catRAPID database (http://www.tartaglialab.com/) to gain insights into their binding specificity. G) RIP assays were employed to quantitatively determine the enrichment of circ_0005185 within the 51–102, 101–152, 146–197, and 196–247 amino acid (aa) regions of the OTUB1 protein. H) Similarly, RIP assays were employed to quantitatively assess the enrichment of circ_0005185 within the 32–83, 51–102, 101–152, and 126–177 aa regions of the RAB8A protein. Data are presented as the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001, ns, not significant).

Article Snippet: Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17‐701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein‐bound RNA, and IgG primary antibody was used as a control.

Techniques: Silver Staining, SDS Page, Isolation, Western Blot, Binding Assay, Immunoprecipitation

Journal: Advanced Science

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway

doi: 10.1002/advs.202411675

Figure Lengend Snippet: circ_0005185 facilitates the deubiquitination of RAB8A by mediating the interaction between OTUB1 and RAB8A. A) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185. B) The Co‐IP experiment used OTUB1 antibody to verify the binding between RAB8A and OTUB1, which increased after overexpression of circ_0005185. C) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185, while knockdown of OTUB1 rescued the level of RAB8A. D) The results of immunofluorescence showed the localization and expression of RAB8A in the control group and circ_0005185 overexpression group. E,F) The ubiquitination level of RAB8A was detected in DU145 and 22RV1 cells using ubiquitination antibodies. Overexpression of circ_0005185 led to decreased ubiquitination of RAB8A, while knockdown of OTUB1 resulted in increased ubiquitination of RAB8A. G,H) The regulation of ubiquitination at the K48 site of RAB8A by OTUB1 was detected in DU145 and 22RV1 cells using antibodies specific to the K48 ubiquitination site. I) Ubiquitination at the K63 site of RAB8A was detected in DU145 cells using antibodies specific to the K63 ubiquitination site.

Techniques: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Knockdown, Immunofluorescence, Expressing, Control, Ubiquitin Proteomics

A. The LCMS/MS detected sequences of OTUB1 in the FLAG-FOXM1 IP complex. B. GST-pulldown showed purified GST-FoxM1 but not GST binding to the purified HIS-OTUB1 protein. C. The IP results suggest reciprocal interactions between endogenous OTUB1 and FOXM1 in CAOV3 cells. 15% of cell lysates were used as input. D. The co-IP results indicate that ectopic FLAG-OTUB1 interacted with GFP-FOXM1 in A2780 and SKOV3 cells. 15% of cell lysates were used as input. E. The IP results of indicate that either knockdown of OTUB1 or FOXM1 by siRNA reduced the binding FOXM1 or OTUB1 in CAOV3 cells. 15% of cell lysates were used as input.

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A. The LCMS/MS detected sequences of OTUB1 in the FLAG-FOXM1 IP complex. B. GST-pulldown showed purified GST-FoxM1 but not GST binding to the purified HIS-OTUB1 protein. C. The IP results suggest reciprocal interactions between endogenous OTUB1 and FOXM1 in CAOV3 cells. 15% of cell lysates were used as input. D. The co-IP results indicate that ectopic FLAG-OTUB1 interacted with GFP-FOXM1 in A2780 and SKOV3 cells. 15% of cell lysates were used as input. E. The IP results of indicate that either knockdown of OTUB1 or FOXM1 by siRNA reduced the binding FOXM1 or OTUB1 in CAOV3 cells. 15% of cell lysates were used as input.

Article Snippet: Consecutive paraffin sections of the 200 cases were prepared and incubated overnight at 37°C with primary antibodies against OTUB1 (HPA039176, Sigma Aldrich, USA) and FOXM1 (K-19, Santa Cruz) at a 1:50 dilution.

Techniques: Purification, Binding Assay, Co-Immunoprecipitation Assay

A. Representative images of score 0-3 of OTUB1 and FOXM1 expression detected by immunochemistry. B. Pearson correlation analysis of immunostaining scores of OTUB1 and FOXM1 ( r =0.610, p <0.01). C. IB results of OTUB1 and FOXM1 in 25 tissues of ovarian carcinoma at Stage I, II and III. D. RT-qPCR results of OTUB1 and FOXM1 in 25 tissues of ovarian carcinoma at Stage I, II and III. E. Kaplan-Meier disease-free survival (DFS) curves of patients with different expressions of OTUB1 in ovarian cancer (Low vs. High). F. Kaplan-Meier disease-specific survival (DSS) curves of patients with different expressions of OTUB1 in ovarian cancer (Low vs. High).

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A. Representative images of score 0-3 of OTUB1 and FOXM1 expression detected by immunochemistry. B. Pearson correlation analysis of immunostaining scores of OTUB1 and FOXM1 ( r =0.610, p <0.01). C. IB results of OTUB1 and FOXM1 in 25 tissues of ovarian carcinoma at Stage I, II and III. D. RT-qPCR results of OTUB1 and FOXM1 in 25 tissues of ovarian carcinoma at Stage I, II and III. E. Kaplan-Meier disease-free survival (DFS) curves of patients with different expressions of OTUB1 in ovarian cancer (Low vs. High). F. Kaplan-Meier disease-specific survival (DSS) curves of patients with different expressions of OTUB1 in ovarian cancer (Low vs. High).

Techniques: Expressing, Immunostaining, Quantitative RT-PCR

Correlation between clinicopathological features and expression of OTUB1

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: Correlation between clinicopathological features and expression of OTUB1

Techniques: Expressing

$A. Immunoblotting results of FOXM1 and OTUB1 in A2780 and SKOV3 cells transiently transfected with indicated plasmids and in CAOV3 cells with siOTUB1. B. RT-qPCR results of FOXM1 and OTUB1 in A2780 and SKOV3 cells transiently transfected with indicated plasmids and in CAOV3 cells with siOTUB1. C. CAOV3 cells were transfected with scramble or siOTUB1 for 48 h. MG132 (10 μM) was added for 3 hours before harvesting. Lysates were immunoblotted with indicated antibodies. D. A2780 and SKOV3 cells were transfected with indicated plasmids for 48 h. CHX (50 μg/mL) then was added at 0 h, 1 h, 2 h, 4 h, and 6 h. Lysates were collected at indicated time points and immunoblotted with indicated antibodies. E. The immunofluorescence results of cellular localization of FOXM1 and FLAG-OTUB1 in A2780 and SKOV3 cells. F. The western blot results of nuclear/cytoplasm fractioned FOXM1 protein levels in OTUB1-overexpressing A2780 and SKOV3 cells.$

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A. Immunoblotting results of FOXM1 and OTUB1 in A2780 and SKOV3 cells transiently transfected with indicated plasmids and in CAOV3 cells with siOTUB1. B. RT-qPCR results of FOXM1 and OTUB1 in A2780 and SKOV3 cells transiently transfected with indicated plasmids and in CAOV3 cells with siOTUB1. C. CAOV3 cells were transfected with scramble or siOTUB1 for 48 h. MG132 (10 μM) was added for 3 hours before harvesting. Lysates were immunoblotted with indicated antibodies. D. A2780 and SKOV3 cells were transfected with indicated plasmids for 48 h. CHX (50 μg/mL) then was added at 0 h, 1 h, 2 h, 4 h, and 6 h. Lysates were collected at indicated time points and immunoblotted with indicated antibodies. E. The immunofluorescence results of cellular localization of FOXM1 and FLAG-OTUB1 in A2780 and SKOV3 cells. F. The western blot results of nuclear/cytoplasm fractioned FOXM1 protein levels in OTUB1-overexpressing A2780 and SKOV3 cells.

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Immunofluorescence

A-B. The in vivo ubiquitination results show that overexpression of FLAG-OTUB1 suppressed the ubiquitination of endogenous FOXM1 in CAOV3 (A) and exogenous MYC-FOXM1 in SKOV3 cells (B). C. The in vitro deubiquitination results that the ubiquitinated FLAG-FOXM1 was deubiquitinated in vitro by purified HIS-OTUB1. D. Lysates of CAOV3 cells were subjected to IP with anti-K48, anti-K63 or anti-K11 antibodies followed by IB with anti-FOXM1 antibody. 15% of cell lysates were used as input. E. The in vitro deubiquitination assays showed that the K48-specific other than K-11 or K-63 ubiquitinated FOXM1 was deubiquitinated in vitro by purified HIS-OTUB1.

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A-B. The in vivo ubiquitination results show that overexpression of FLAG-OTUB1 suppressed the ubiquitination of endogenous FOXM1 in CAOV3 (A) and exogenous MYC-FOXM1 in SKOV3 cells (B). C. The in vitro deubiquitination results that the ubiquitinated FLAG-FOXM1 was deubiquitinated in vitro by purified HIS-OTUB1. D. Lysates of CAOV3 cells were subjected to IP with anti-K48, anti-K63 or anti-K11 antibodies followed by IB with anti-FOXM1 antibody. 15% of cell lysates were used as input. E. The in vitro deubiquitination assays showed that the K48-specific other than K-11 or K-63 ubiquitinated FOXM1 was deubiquitinated in vitro by purified HIS-OTUB1.

Techniques: In Vivo, Over Expression, In Vitro, Purification

A. The structure of full-length OTUB1 and its mutants. The key residues and domains of OTUB1 were shown. B. The GST-pulldown shows that the N-terminal 16-46aa and C-terminus of OTUB1 bound to GST-FOXM1 in vitro. C. The co-IP shows that the N-terminal 16-46aa and C-terminus of OTUB1 interact with FOXM1 in vivo. 15% of cell lysates were used as input. D. The in vitro ubiquitination assay shows that the DUB activity on FOXM1 was attenuated in both OTUB1 ΔN46 and OTUB1 A/S/A . E. The in vivo ubiquitination results show that the polyubiquitination of FOXM1 MutKEN and FOXM1 ΔN30/MutKEN was reduced compared with full-length FOXM1 and FOXM1 ΔN30 in H293T cells. F. The co-IP results indicate that the affinities of FOXM1 MutKEN and FOXM1 ΔN30/MutKEN for OTUB1 were attenuated compared with full-length FOXM1 and FOXM1 ΔN30 in SKOV3 cells. 15% of cell lysates were used as input. OTUB1 A/S/A : OTUB1 D88A/C91S/H265A .

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A. The structure of full-length OTUB1 and its mutants. The key residues and domains of OTUB1 were shown. B. The GST-pulldown shows that the N-terminal 16-46aa and C-terminus of OTUB1 bound to GST-FOXM1 in vitro. C. The co-IP shows that the N-terminal 16-46aa and C-terminus of OTUB1 interact with FOXM1 in vivo. 15% of cell lysates were used as input. D. The in vitro ubiquitination assay shows that the DUB activity on FOXM1 was attenuated in both OTUB1 ΔN46 and OTUB1 A/S/A . E. The in vivo ubiquitination results show that the polyubiquitination of FOXM1 MutKEN and FOXM1 ΔN30/MutKEN was reduced compared with full-length FOXM1 and FOXM1 ΔN30 in H293T cells. F. The co-IP results indicate that the affinities of FOXM1 MutKEN and FOXM1 ΔN30/MutKEN for OTUB1 were attenuated compared with full-length FOXM1 and FOXM1 ΔN30 in SKOV3 cells. 15% of cell lysates were used as input. OTUB1 A/S/A : OTUB1 D88A/C91S/H265A .

Techniques: In Vitro, Co-Immunoprecipitation Assay, In Vivo, Ubiquitin Assay, Activity Assay

A. The western blotting results showed the expression of downstream targets of FOXM1 SNAIL, CDC25B and CyclinB in NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. B. The CCK8 results of NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. *: p <0.01. C. Representative images of EdU immunofluorescence and transwell assay in NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. The percentage of EdU positive cells were graphed under 100× and calculated under 200× magnification. The penetrated cells were graphed under 200× and counted under 400× magnification. *: p <0.01. **: p <0.05. D. The xenograft tumorigenesis results of NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. Tumors were photographed and the speed of tumor growth was illustrated as curves, and the weights of tumors were calculated and analyzed. *: p <0.01. **: p <0.05.

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A. The western blotting results showed the expression of downstream targets of FOXM1 SNAIL, CDC25B and CyclinB in NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. B. The CCK8 results of NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. *: p <0.01. C. Representative images of EdU immunofluorescence and transwell assay in NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. The percentage of EdU positive cells were graphed under 100× and calculated under 200× magnification. The penetrated cells were graphed under 200× and counted under 400× magnification. *: p <0.01. **: p <0.05. D. The xenograft tumorigenesis results of NC, OTUB1, OTUB1-shC and OTUB1-shFOXM1 infected SKOV3 cells. Tumors were photographed and the speed of tumor growth was illustrated as curves, and the weights of tumors were calculated and analyzed. *: p <0.01. **: p <0.05.

Techniques: Western Blot, Expressing, Infection, Immunofluorescence, Transwell Assay

A. Representative images of EdU immunofluorescence and transwell assay in NC, OTUB1, OTUB1 ΔN46 and OTUB1 A/S/A infected SKOV3 cells. The percentage of EdU positive cells were graphed under 100× and calculated under 200× magnification. The penetrated cells were graphed under 200× and counted under 400× magnification. *: p <0.01. **: p <0.05. B-C. The xenograft tumorigenesis results of NC, OTUB1, OTUB1 ΔN46 and OTUB1 A/S/A infected SKOV3 cells. Tumors were photographed (B) and the speed of tumor growth was illustrated as curves, and the weights of tumors were calculated and analyzed (C). *: p <0.01. D. Representative images of EdU immunofluorescence and transwell assay in NC, OTUB1, FOXM1, OTUB1-NC(FOXM1), OTUB1-FOXM1 and OTUB1-FOXM1 MutKEN infected SKOV3 cells. The percentage of EdU positive cells were graphed under 100× and calculated under 200× magnification. The penetrated cells were graphed under 200× and counted under 400× magnification. *: p <0.01. E-F. The xenograft tumorigenesis results of NC, OTUB1, FOXM1, OTUB1-NC(FOXM1), OTUB1-FOXM1, and OTUB1-FOXM1 MutKEN infected SKOV3 cells (E). Tumors were photographed and the speed of tumor growth was illustrated as curves, and the weights of tumors were calculated and analyzed (F). *: p <0.01. FOXM1 MutKEN : FOXM1 K207A/E208A/N209A .

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: A. Representative images of EdU immunofluorescence and transwell assay in NC, OTUB1, OTUB1 ΔN46 and OTUB1 A/S/A infected SKOV3 cells. The percentage of EdU positive cells were graphed under 100× and calculated under 200× magnification. The penetrated cells were graphed under 200× and counted under 400× magnification. *: p <0.01. **: p <0.05. B-C. The xenograft tumorigenesis results of NC, OTUB1, OTUB1 ΔN46 and OTUB1 A/S/A infected SKOV3 cells. Tumors were photographed (B) and the speed of tumor growth was illustrated as curves, and the weights of tumors were calculated and analyzed (C). *: p <0.01. D. Representative images of EdU immunofluorescence and transwell assay in NC, OTUB1, FOXM1, OTUB1-NC(FOXM1), OTUB1-FOXM1 and OTUB1-FOXM1 MutKEN infected SKOV3 cells. The percentage of EdU positive cells were graphed under 100× and calculated under 200× magnification. The penetrated cells were graphed under 200× and counted under 400× magnification. *: p <0.01. E-F. The xenograft tumorigenesis results of NC, OTUB1, FOXM1, OTUB1-NC(FOXM1), OTUB1-FOXM1, and OTUB1-FOXM1 MutKEN infected SKOV3 cells (E). Tumors were photographed and the speed of tumor growth was illustrated as curves, and the weights of tumors were calculated and analyzed (F). *: p <0.01. FOXM1 MutKEN : FOXM1 K207A/E208A/N209A .

Techniques: Immunofluorescence, Transwell Assay, Infection

FOXM1 is ubiquitinated by polyUb chains in cytoplasm leading to a proteasome-mediated degradation, which needs the conformation of its N-terminal 30aa and KEN box. OTUB1 is recruited and binds to FOXM1 and then recognizes the K48-specific linkage. Once confirming the linkage, OTUB1 binds to Ub via both the N-terminal proximal Ub binding site (N-terminal 16-46aa) and the OTU domain, cleaving the linkage and stabilize FOXM1. Consequently, more FOXM1 enters the nucleus to exert its function to activate the transcription of downstream genes.

Journal: Oncotarget

Article Title: OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9160

Figure Lengend Snippet: FOXM1 is ubiquitinated by polyUb chains in cytoplasm leading to a proteasome-mediated degradation, which needs the conformation of its N-terminal 30aa and KEN box. OTUB1 is recruited and binds to FOXM1 and then recognizes the K48-specific linkage. Once confirming the linkage, OTUB1 binds to Ub via both the N-terminal proximal Ub binding site (N-terminal 16-46aa) and the OTU domain, cleaving the linkage and stabilize FOXM1. Consequently, more FOXM1 enters the nucleus to exert its function to activate the transcription of downstream genes.

Techniques: Binding Assay

Review

otub1 primary antibody (Proteintech)

Structured Review

Images

1) Product Images from "Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway"

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