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.

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

Journal: International Journal of Molecular Medicine

Article Title: Transcription factor EB-mediated autophagy promotes dermal fibroblast differentiation and collagen production by regulating endoplasmic reticulum stress and autophagy-dependent secretion

doi: 10.3892/ijmm.2020.4814

Figure Lengend Snippet: TGF-β1-induced COL I secretion from fibroblasts is associated with an autophagy-based pathway. Fibroblasts were treated with 50 nmol/l TFEB-siRNA or NC-siRNA for 48 h and then with 10 ng/ml TGF-β1 for 48 h. (A) Fluorescence for colocalization of Rab8a with LAMP1 and COL I (scale bars, 10 or 30 µ m). (B) Line tracing analysis of fluorescence signal intensity. (C) Pearson's colocalization coefficient for COL I and LAMP1. (D) Pearson's colocalization coefficient for COL I and Rab8a. (E) Pro-COL Iα1 secretion in the supernatant of fibroblasts was assessed by enzyme-linked immunosorbent assay. Results are presented as the mean ± SD (n=6). ** P<0.01 vs. control group; ## P<0.01 vs. TGF-β1 treatment group. TGF-β1, transforming growth factor-β1; TFEB, transcription factor EB; siRNA, small interfering RNA; NC, negative control; COL I, collagen I; Rab8a, Ras-related protein Rab-8A; LAMP1, lyso-some-associated membrane protein 1; ns, not significant compared with TGF-β1 treatment group.

Article Snippet: Subsequently, cells were incubated with the following primary antibodies overnight at 4°C: Anti-TFEB (1:50; cat. no. ab220695; Abcam), anti-LAMP1 (1:200; cat. no. ab62562; Abcam), anti-COL I (1:200; cat. no. ab6308; Abcam) and anti-Ras-related protein Rab-8A (Rab8a; 1:200; cat. no. ABIN6290618; Beijing 4A Biotech Co., Ltd.).

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay, Small Interfering RNA, Negative Control