diluted anti-agps antibody  (Servicebio Inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

doi: 10.1186/s13046-023-02920-w

Figure Lengend Snippet: TrkA promotes the ubiquitinated degradation of AGPS by MDM2 by modifying the phosphorylation of AGPS. a Western blot analysis of endogenous AGPS proteins in DU145 and 22Rv1 cells followed by treatment with different phosphatase inhibitors (Merestinib, 10 μM for 24 h; AG1295,10 μM for 2 h; AIM-100, 10 μM overnight; Dacomitinib, 2 μM for 24 h; Larotrectinib, 20 nM overnight). b Co-IP analysis of the phosphorylation levels of Flag-tagged AGPS in DU145 cells after being treated with MG132 (20 μM) for 8 h and Larotrectinib. c Western blot analysis of TrkA and p-TrkA expression in DU145, 22Rv1 and RWPE-1 cells. d Analysis of correlation between IHC staining of AGPS and p-TrkA proteins in different Gleason score PCa patient specimens. e Western blot analysis of AGPS protein expression in DU145 and 22Rv1 cells with or without Larotrectinib treatment. f Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with Larotrectinib. g Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with MG132 (20 μM) for 8 h or with Larotrectinib. h Western blot analysis of AGPS protein expression in DU145 cells with or without CHX (50 μg/mL) treatment for 0-, 4-, 8-, 12-, 16-, or 20- h. i Protein bands were quantified and normalized to the band intensity at the 0-h time point. * P < 0.05, ** P < 0.01, *** P < 0.001. j Co-IP analysis of binding of Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells after NTRK1 knockdown. k Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells after NTRK1 knockdown and treated with MG132 (20 μM) for 8 h

Article Snippet: These sections were dyed with diluted anti-AGPS and anti-p-TrkA antibody and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature.

Techniques: Phospho-proteomics, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunohistochemistry, Ubiquitin Proteomics, Binding Assay, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

doi: 10.1186/s13046-023-02920-w

Figure Lengend Snippet: Larotrectinib promotes the sensitivity of prostate cancer cells to ferroptosis by inhibiting the phosphorylation of TrkA. a Western blot analysis of AGPS protein expression in DU145 and 22Rv1 cells with or without ML210 (10 μM for 8 h) and Larotrectinib (20 nM, overnight) treatment. b The sensitive of ferroptosis in DU145 and 22Rv1 cells with or without ML210 and Larotrectinib treatment. ** P < 0.01, *** P < 0.001. c , d PMP70 staining in DU145 and 22Rv1 cells with or without ML210 and Larotrectinib treatment. ** P < 0.01, *** P < 0.001, **** P < 0.0001. e , f Pca cells were observed by TEM with or without ML210 and Larotrectinib treatment. ** P < 0.01, *** P < 0.001, **** P < 0.0001. g MDA level after treatment with ML210 and/or Larotrectinib in DU145 and 22Rv1 cells. * P < 0.05, ** P < 0.01. h CCK8 essay with the OD values in 6-wells plate after treatment with ML210 and/or Larotrectinib in DU145 and 22Rv1 cells. ** P < 0.01, *** P < 0.001, **** P < 0.0001. i Colony formation assay displayed the DU145 and 22Rv1 cell colony numbers after treatment with ML210 and/or Larotrectinib. j Organoid culture treated with ML210 and/or Larotrectinib from Pca patient tissues. k PCa cells were injected subcutaneously into the right flank of mice after treatment with ML210(50 mg/kg) and/or Larotrectinib (300 mg/kg) every other 24 h. Xenograft growth was measured every other day for 28 days. Tumors in each group at day 28 were harvested and photographed. l Tumor weight in different groups. Data represented as mean ± SD ( n = 5). ** P < 0.01, **** P < 0.0001. m Tumor volume at each time point. Data represented as mean ± SD ( n = 5). n.s., no significance; * P < 0.01, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: These sections were dyed with diluted anti-AGPS and anti-p-TrkA antibody and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature.

Techniques: Phospho-proteomics, Western Blot, Expressing, Staining, Colony Assay, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

doi: 10.1186/s13046-023-02920-w

Figure Lengend Snippet: A hypothetic model depicting the dual regulation mechanism of AGPS protein degradation by MDM2 and TrkA inhibitor. MDM2 ubiquitinates and promotes the degradation of AGPS, phosphorylation kinase TrkA aberrantly activated in prostate cancer and facilitates MDM2 ubiquitinate AGPS. TrkA inhibitor Larotrectinib could reactivate peroxisome pathway and sensitive prostate cancer cells to ML210. The combined administration of ML210 and Larotrectinib could significantly ablate the progression of prostate cancer.The model was created by Pro Procreate software and BioRender website ( www.biorender.com )

Article Snippet: These sections were dyed with diluted anti-AGPS and anti-p-TrkA antibody and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature.

Techniques: Phospho-proteomics, Software