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rabbit polyclonal usp10 antibody  (Bethyl)


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    Structured Review

    Bethyl rabbit polyclonal usp10 antibody
    Chronic stress-pre-incubated cells fail to disassemble polysomes by acute stress U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 1 μM Tg (thapsigargin), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (A) Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, G3BP1, G3BP2, caprin1, <t>USP10,</t> and β-actin. Three blots were taken for each of the three biological replicates (independent experiments). The same colors of the circle dot in the graph are from the same independent experiment. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗ <0.05, p ∗∗ <0.01, p ∗∗∗ <0.001, p ∗∗∗∗ <0.0001). (B–F) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; (B) 24 h pre-incubation of SA, blue; 1 h SA with 24 h pre-incubation of SA, green. (C) 24 h pre-incubation of Tg, blue; 1 h SA with 24 h pre-incubation of Tg, green. (D) 24 h pre-incubation of CCCP, blue; 1 h SA with 24 h pre-incubation of CCCP; green. (E) 24 h pre-incubation of HBSS, blue; 1 h SA with 24 h pre-incubation of HBSS; green. (F) The polysome/monosome ratio was calculated with normalization by each of NT as a control.
    Rabbit Polyclonal Usp10 Antibody, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/usp10/USP10+Antibody/pmc12828549-18-0-5
    Average 93 stars, based on 35 article reviews
    rabbit polyclonal usp10 antibody - by Bioz Stars, 2026-10
    93/100 stars

    Images

    1) Product Images from "Chronic stress antagonizes formation of stress granules"

    Article Title: Chronic stress antagonizes formation of stress granules

    Journal: iScience

    doi: 10.1016/j.isci.2025.114556

    Chronic stress-pre-incubated cells fail to disassemble polysomes by acute stress U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 1 μM Tg (thapsigargin), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (A) Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, G3BP1, G3BP2, caprin1, USP10, and β-actin. Three blots were taken for each of the three biological replicates (independent experiments). The same colors of the circle dot in the graph are from the same independent experiment. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗ <0.05, p ∗∗ <0.01, p ∗∗∗ <0.001, p ∗∗∗∗ <0.0001). (B–F) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; (B) 24 h pre-incubation of SA, blue; 1 h SA with 24 h pre-incubation of SA, green. (C) 24 h pre-incubation of Tg, blue; 1 h SA with 24 h pre-incubation of Tg, green. (D) 24 h pre-incubation of CCCP, blue; 1 h SA with 24 h pre-incubation of CCCP; green. (E) 24 h pre-incubation of HBSS, blue; 1 h SA with 24 h pre-incubation of HBSS; green. (F) The polysome/monosome ratio was calculated with normalization by each of NT as a control.
    Figure Legend Snippet: Chronic stress-pre-incubated cells fail to disassemble polysomes by acute stress U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 1 μM Tg (thapsigargin), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (A) Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, G3BP1, G3BP2, caprin1, USP10, and β-actin. Three blots were taken for each of the three biological replicates (independent experiments). The same colors of the circle dot in the graph are from the same independent experiment. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗ <0.05, p ∗∗ <0.01, p ∗∗∗ <0.001, p ∗∗∗∗ <0.0001). (B–F) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; (B) 24 h pre-incubation of SA, blue; 1 h SA with 24 h pre-incubation of SA, green. (C) 24 h pre-incubation of Tg, blue; 1 h SA with 24 h pre-incubation of Tg, green. (D) 24 h pre-incubation of CCCP, blue; 1 h SA with 24 h pre-incubation of CCCP; green. (E) 24 h pre-incubation of HBSS, blue; 1 h SA with 24 h pre-incubation of HBSS; green. (F) The polysome/monosome ratio was calculated with normalization by each of NT as a control.

    Techniques Used: Incubation, Control, Western Blot

    Related Articles

    Microarray:

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    Article Snippet: Immunostaining for Ub-H2A (D27C4) (Cell Signaling) was performed using a 1/200 dilution of mouse monoclonal and the MOM Peroxidase Kit (Vector Laboratories) following the manufacturer’s instructions and as we previously described . .. Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described . ..

    Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.
    Article Snippet: .. Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68. ..

    Immunostaining:

    Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells
    Article Snippet: Immunostaining for Ub-H2A (D27C4) (Cell Signaling) was performed using a 1/200 dilution of mouse monoclonal and the MOM Peroxidase Kit (Vector Laboratories) following the manufacturer’s instructions and as we previously described . .. Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described . ..

    Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.
    Article Snippet: .. Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68. ..

    Immunohistochemistry:

    Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells
    Article Snippet: Immunostaining for Ub-H2A (D27C4) (Cell Signaling) was performed using a 1/200 dilution of mouse monoclonal and the MOM Peroxidase Kit (Vector Laboratories) following the manufacturer’s instructions and as we previously described . .. Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described . ..

    Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.
    Article Snippet: .. Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68. ..

    other:

    Article Title: USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B‐cell lymphoma
    Article Snippet: The following antibodies were used: beta‐actin (1:10,000, mouse), BIRC8 (mouse, Abnova # H00112401‐B01), cIAP1/2, cleaved caspase‐3‐5A1E (1:300, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32‐2400), cyclin A (1:1,000, mouse, Santa Cruz #sc‐751), cyclin B1 (1:1,000, mouse, Cell Signaling #4138), cyclin E (1:1,000, mouse, kind gift of M. Pagano), FLAG (1:1,000, rabbit, Sigma #F7425), FLAG‐M2 (1:1,000, mouse, Sigma #F3165), HA‐16B12 (1:2,000, mouse, Covance #MMS‐101P), human MCL1 (1:500, rabbit, BD Pharmingen #554103), murine MCL1 (1:1,000, rabbit, A&D Serotec AHP1249), ML‐IAP (mouse, Santa Cruz #sc‐71592), NIAP (rabbit, Abcam #ab25968), PARP1/2 (1:1,000, rabbit, Santa Cruz #sc‐7150), pHH3 (S10) (1:300, rabbit, Cell Signaling #9701), PLK1 (1:500, rabbit, Invitrogen #33‐1700), survivin (1:3,000, rabbit, R&D Systems #AF886), USP7 (rabbit, Bethyl Lab. #A300‐033A), USP9X (1:4,000, rabbit, Bethyl Laboratories #A301‐351A), USP10 (rabbit, Bethyl Lab. #A300‐900A), USP24 (Proteintech Europe, #13126‐1‐AP), USP28 (rabbit, Bethyl Lab. #A300‐898A), V5 (1:1,000, rabbit, Sigma #V8137), XIAP (1:1,000, mouse, BD Biosciences #610716), XIAP (1:1,000, rabbit, Cell Signaling #2042), and XIAP (1:1,000, R&D Systems #AF8221).

    Western Blot:

    Article Title: A functional interaction between TDP-43 and USP10 reveals USP10 dysfunction in TDP-43 proteinopathies
    Article Snippet: All constructs were amplified in commercial E. coli lines and plasmids purified (740410.50, Macherey Nagel) according to the manufacturer’s instructions. .. The following antibodies and concentrations were used for immunoblot and immunofluorescence staining: Rabbit anti TDP-43 (WB 1:1000, IF 1:400, Protein Tech, 10782-2-AP); mouse anti TDP-43 (WB 1:5000, abcam, ab104223); mouse anti TDP-43 (IF 1:200, R&D systems, MAB37778); mouse anti pTDP-43 (IF 1:3000, Cosmobio, CAC-TIP-PTD-M01); mouse anti G3BP1 (IF 1:300, Protein Tech, 66486-1-Ig); rabbit anti G3BP1 (WB: 1:2000, IF 1:300, Protein Tech, 13057-2-AP); rabbit anti G3BP2 (IF 1:300, Protein Tech, 16276-1-AP); rabbit anti USP10 (WB 1:1000, IF 1:500, Sigma-Aldrich, HPA006731); rabbit anti USP10 (WB 1:1000, made in house from the Freire laboratory); rabbit anti USP10 (IF 1:200, Bethyl, A300-901A); mouse anti p53 (WB 1:500, Santa Cruz Biotechnology, sc-126); mouse anti Tubulin (WB 1:10000, Protein Tech, 11224-1-AP); mouse anti β-Tubulin (WB 1:10000, Protein Tech, 66240-1-Ig); mouse anti GAPDH (WB 1:250, Santa Cruz Biotechnology, sc-32233); anti mouse Alexa Fluor 488 (IF secondary 1:600, Thermo Fisher, Z25002); anti rabbit Alexa Fluor 488 (IF secondary 1:600, Thermo Fisher, Z25302); anti mouse Alexa Fluor 568 (IF secondary 1:600, Thermo Fisher, Z25006); anti rabbit Alexa Fluor 568 (IF secondary 1:600, Thermo Fisher, Z25306). ..

    Article Title: Thymosin α1 represents a potential potent single molecule-based therapy for cystic fibrosis
    Article Snippet: .. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME DUB complex(s) using an anti-HA antibody (A190-108A, Bethyl Laboratories) followed by SDS-PAGE and Western blot analysis using specific anti-DUB antibodies USP19 (Bethyl Laboratories), USP10 (Bethyl Laboratories) and USP36 (Proteintech). ..

    Immunofluorescence:

    Article Title: A functional interaction between TDP-43 and USP10 reveals USP10 dysfunction in TDP-43 proteinopathies
    Article Snippet: All constructs were amplified in commercial E. coli lines and plasmids purified (740410.50, Macherey Nagel) according to the manufacturer’s instructions. .. The following antibodies and concentrations were used for immunoblot and immunofluorescence staining: Rabbit anti TDP-43 (WB 1:1000, IF 1:400, Protein Tech, 10782-2-AP); mouse anti TDP-43 (WB 1:5000, abcam, ab104223); mouse anti TDP-43 (IF 1:200, R&D systems, MAB37778); mouse anti pTDP-43 (IF 1:3000, Cosmobio, CAC-TIP-PTD-M01); mouse anti G3BP1 (IF 1:300, Protein Tech, 66486-1-Ig); rabbit anti G3BP1 (WB: 1:2000, IF 1:300, Protein Tech, 13057-2-AP); rabbit anti G3BP2 (IF 1:300, Protein Tech, 16276-1-AP); rabbit anti USP10 (WB 1:1000, IF 1:500, Sigma-Aldrich, HPA006731); rabbit anti USP10 (WB 1:1000, made in house from the Freire laboratory); rabbit anti USP10 (IF 1:200, Bethyl, A300-901A); mouse anti p53 (WB 1:500, Santa Cruz Biotechnology, sc-126); mouse anti Tubulin (WB 1:10000, Protein Tech, 11224-1-AP); mouse anti β-Tubulin (WB 1:10000, Protein Tech, 66240-1-Ig); mouse anti GAPDH (WB 1:250, Santa Cruz Biotechnology, sc-32233); anti mouse Alexa Fluor 488 (IF secondary 1:600, Thermo Fisher, Z25002); anti rabbit Alexa Fluor 488 (IF secondary 1:600, Thermo Fisher, Z25302); anti mouse Alexa Fluor 568 (IF secondary 1:600, Thermo Fisher, Z25006); anti rabbit Alexa Fluor 568 (IF secondary 1:600, Thermo Fisher, Z25306). ..

    Staining:

    Article Title: A functional interaction between TDP-43 and USP10 reveals USP10 dysfunction in TDP-43 proteinopathies
    Article Snippet: All constructs were amplified in commercial E. coli lines and plasmids purified (740410.50, Macherey Nagel) according to the manufacturer’s instructions. .. The following antibodies and concentrations were used for immunoblot and immunofluorescence staining: Rabbit anti TDP-43 (WB 1:1000, IF 1:400, Protein Tech, 10782-2-AP); mouse anti TDP-43 (WB 1:5000, abcam, ab104223); mouse anti TDP-43 (IF 1:200, R&D systems, MAB37778); mouse anti pTDP-43 (IF 1:3000, Cosmobio, CAC-TIP-PTD-M01); mouse anti G3BP1 (IF 1:300, Protein Tech, 66486-1-Ig); rabbit anti G3BP1 (WB: 1:2000, IF 1:300, Protein Tech, 13057-2-AP); rabbit anti G3BP2 (IF 1:300, Protein Tech, 16276-1-AP); rabbit anti USP10 (WB 1:1000, IF 1:500, Sigma-Aldrich, HPA006731); rabbit anti USP10 (WB 1:1000, made in house from the Freire laboratory); rabbit anti USP10 (IF 1:200, Bethyl, A300-901A); mouse anti p53 (WB 1:500, Santa Cruz Biotechnology, sc-126); mouse anti Tubulin (WB 1:10000, Protein Tech, 11224-1-AP); mouse anti β-Tubulin (WB 1:10000, Protein Tech, 66240-1-Ig); mouse anti GAPDH (WB 1:250, Santa Cruz Biotechnology, sc-32233); anti mouse Alexa Fluor 488 (IF secondary 1:600, Thermo Fisher, Z25002); anti rabbit Alexa Fluor 488 (IF secondary 1:600, Thermo Fisher, Z25302); anti mouse Alexa Fluor 568 (IF secondary 1:600, Thermo Fisher, Z25006); anti rabbit Alexa Fluor 568 (IF secondary 1:600, Thermo Fisher, Z25306). ..

    Immunoprecipitation:

    Article Title: Thymosin α1 represents a potential potent single molecule-based therapy for cystic fibrosis
    Article Snippet: .. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME DUB complex(s) using an anti-HA antibody (A190-108A, Bethyl Laboratories) followed by SDS-PAGE and Western blot analysis using specific anti-DUB antibodies USP19 (Bethyl Laboratories), USP10 (Bethyl Laboratories) and USP36 (Proteintech). ..

    SDS Page:

    Article Title: Thymosin α1 represents a potential potent single molecule-based therapy for cystic fibrosis
    Article Snippet: .. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME DUB complex(s) using an anti-HA antibody (A190-108A, Bethyl Laboratories) followed by SDS-PAGE and Western blot analysis using specific anti-DUB antibodies USP19 (Bethyl Laboratories), USP10 (Bethyl Laboratories) and USP36 (Proteintech). ..



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    93
    Bethyl a300 900a rrid ab 625312
    Chronic stress-pre-incubated cells fail to disassemble polysomes by acute stress U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 1 μM Tg (thapsigargin), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (A) Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, G3BP1, G3BP2, caprin1, <t>USP10,</t> and β-actin. Three blots were taken for each of the three biological replicates (independent experiments). The same colors of the circle dot in the graph are from the same independent experiment. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗ <0.05, p ∗∗ <0.01, p ∗∗∗ <0.001, p ∗∗∗∗ <0.0001). (B–F) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; (B) 24 h pre-incubation of SA, blue; 1 h SA with 24 h pre-incubation of SA, green. (C) 24 h pre-incubation of Tg, blue; 1 h SA with 24 h pre-incubation of Tg, green. (D) 24 h pre-incubation of CCCP, blue; 1 h SA with 24 h pre-incubation of CCCP; green. (E) 24 h pre-incubation of HBSS, blue; 1 h SA with 24 h pre-incubation of HBSS; green. (F) The polysome/monosome ratio was calculated with normalization by each of NT as a control.
    A300 900a Rrid Ab 625312, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CircAFF4 directly binds to the deubiquitinase USP10. A Coomassie brilliant blue staining of proteins pulled down by biotin-labeled probes specific to circAFF4 and control probes. B venn diagram showing ubiquitin-proteasome system proteins from the uniprot database and proteins detected by mass spectrometry (MS). C Western blot showing that USP10 was captured by the circAFF4 probe in the RNA pull-down assay. D immunofluorescence-FISH was used to detect the co-localization of circAFF4 and USP10 in the cytoplasm. E RT-qPCR detection of RNA samples obtained from RIP assay showed that circAFF4 was captured by USP10. F RIP assay was performed in A549 cells using anti-USP10 antibody and IgG control under specified conditions, followed by qRT-PCR. G CircAFF4 binds to USP10 proteins via CatRAPID prediction. H schematic diagram of full-length and truncated USP10 proteins. I RIP assays were performed using anti-Flag antibody in A549 cells transfected with the specified full-length or truncated USP10 plasmids (containing 3×Flag tag). The co-precipitated proteins and RNA were purified, followed by Western blot and RT-qPCR, respectively. J RIP assay was performed in A549 cells using anti-USP10 antibody and IgG control under specified conditions, followed by qRT-PCR. Data are shown as mean ± SD, n = 3. *** p < 0.01, * p < 0.001

    Journal: Biology Direct

    Article Title: CircAFF4 inhibits lung cancer progression via destabilizing GPX4 and triggering ferroptosis

    doi: 10.1186/s13062-026-00782-8

    Figure Lengend Snippet: CircAFF4 directly binds to the deubiquitinase USP10. A Coomassie brilliant blue staining of proteins pulled down by biotin-labeled probes specific to circAFF4 and control probes. B venn diagram showing ubiquitin-proteasome system proteins from the uniprot database and proteins detected by mass spectrometry (MS). C Western blot showing that USP10 was captured by the circAFF4 probe in the RNA pull-down assay. D immunofluorescence-FISH was used to detect the co-localization of circAFF4 and USP10 in the cytoplasm. E RT-qPCR detection of RNA samples obtained from RIP assay showed that circAFF4 was captured by USP10. F RIP assay was performed in A549 cells using anti-USP10 antibody and IgG control under specified conditions, followed by qRT-PCR. G CircAFF4 binds to USP10 proteins via CatRAPID prediction. H schematic diagram of full-length and truncated USP10 proteins. I RIP assays were performed using anti-Flag antibody in A549 cells transfected with the specified full-length or truncated USP10 plasmids (containing 3×Flag tag). The co-precipitated proteins and RNA were purified, followed by Western blot and RT-qPCR, respectively. J RIP assay was performed in A549 cells using anti-USP10 antibody and IgG control under specified conditions, followed by qRT-PCR. Data are shown as mean ± SD, n = 3. *** p < 0.01, * p < 0.001

    Article Snippet: The membrane was incubated overnight at 4 °C with primary antibodies against GPX4 (Abmart, T56959F), USP10 (SAB, 49,700), SLC7A11 (ABclonal, A2413), SLC3A2 (ABclonal, A3658), ACSL4 (Wanleibio, WL06242), and GAPDH (ZSGB-BIO, TA-08, AB_2747414).

    Techniques: Staining, Labeling, Control, Ubiquitin Proteomics, Mass Spectrometry, Western Blot, Pull Down Assay, Immunofluorescence, Quantitative RT-PCR, Transfection, Purification

    CircAFF4 inhibits USP10-mediated deubiquitination of GPX4 to promote ferroptosis in lung cancer cells. A Co-IP assays were performed to analyze the direct interaction between USP10 and GPX4 in A549 cells, using anti-USP10 antibody or anti-GPX4 antibody respectively. B Western blot assay was used to analyze the change in GPX4 protein level upon USP10 overexpression. C GPX4 protein was captured by co-immunoprecipitation, and its ubiquitination level was detected by Western blot. D Co-IP assays were performed using anti-GPX4 antibody in A549 cells transfected with the specified plasmids. E the ubiquitination level of GPX4 was detected in A549 cells transfected or co-transfected with the specified vectors. F EdU assay was used to determine the proliferative capacity of A549 cells transfected or co-transfected with the specified vectors. G intracellular ROS levels in cells transfected or co-transfected with the specified vectors were detected by DCFH-DA fluorescent staining. Scale bar: 275 μm. H intracellular MDA levels in cells transfected or co-transfected with the specified vectors after 48 hours. I super-resolution microscopy images showing mitochondrial morphology in cells transfected or co-transfected with the specified vectors. Scale bar: 50 μm. Data are shown as mean ± SD, n = 3. *** p < 0.01, * p < 0.001

    Journal: Biology Direct

    Article Title: CircAFF4 inhibits lung cancer progression via destabilizing GPX4 and triggering ferroptosis

    doi: 10.1186/s13062-026-00782-8

    Figure Lengend Snippet: CircAFF4 inhibits USP10-mediated deubiquitination of GPX4 to promote ferroptosis in lung cancer cells. A Co-IP assays were performed to analyze the direct interaction between USP10 and GPX4 in A549 cells, using anti-USP10 antibody or anti-GPX4 antibody respectively. B Western blot assay was used to analyze the change in GPX4 protein level upon USP10 overexpression. C GPX4 protein was captured by co-immunoprecipitation, and its ubiquitination level was detected by Western blot. D Co-IP assays were performed using anti-GPX4 antibody in A549 cells transfected with the specified plasmids. E the ubiquitination level of GPX4 was detected in A549 cells transfected or co-transfected with the specified vectors. F EdU assay was used to determine the proliferative capacity of A549 cells transfected or co-transfected with the specified vectors. G intracellular ROS levels in cells transfected or co-transfected with the specified vectors were detected by DCFH-DA fluorescent staining. Scale bar: 275 μm. H intracellular MDA levels in cells transfected or co-transfected with the specified vectors after 48 hours. I super-resolution microscopy images showing mitochondrial morphology in cells transfected or co-transfected with the specified vectors. Scale bar: 50 μm. Data are shown as mean ± SD, n = 3. *** p < 0.01, * p < 0.001

    Article Snippet: The membrane was incubated overnight at 4 °C with primary antibodies against GPX4 (Abmart, T56959F), USP10 (SAB, 49,700), SLC7A11 (ABclonal, A2413), SLC3A2 (ABclonal, A3658), ACSL4 (Wanleibio, WL06242), and GAPDH (ZSGB-BIO, TA-08, AB_2747414).

    Techniques: Co-Immunoprecipitation Assay, Western Blot, Over Expression, Immunoprecipitation, Ubiquitin Proteomics, Transfection, EdU Assay, Staining, Super-Resolution Microscopy

    CircAFF4 inhibits USP10-mediated deubiquitination of GPX4 to suppress lung cancer growth in vivo. A images of tumor formation in nude mice injected with A549 cells transfected or co-transfected with specific vectors. B the growth curves of xenografted tumors in nude mice from each group were measured once a week. C images of subcutaneous tumor formation in nude mice at 8 weeks. Mice were injected with A549 cells transfected or co-transfected with specific vectors. D the weights of xenografted tumors in each group were calculated. E Western blot assay was used to detect the protein expression of GPX4 and USP10 in subcutaneous xenografts derived from A549 cells. F , G Immunohistochemistry and immunofluorescence assays were used to detect the expression of GPX4 and USP10 in xenografted tumors. Scale bar: 125 μm. Data are shown as mean ± SD, n = 5. *** p < 0.01, * p < 0.001

    Journal: Biology Direct

    Article Title: CircAFF4 inhibits lung cancer progression via destabilizing GPX4 and triggering ferroptosis

    doi: 10.1186/s13062-026-00782-8

    Figure Lengend Snippet: CircAFF4 inhibits USP10-mediated deubiquitination of GPX4 to suppress lung cancer growth in vivo. A images of tumor formation in nude mice injected with A549 cells transfected or co-transfected with specific vectors. B the growth curves of xenografted tumors in nude mice from each group were measured once a week. C images of subcutaneous tumor formation in nude mice at 8 weeks. Mice were injected with A549 cells transfected or co-transfected with specific vectors. D the weights of xenografted tumors in each group were calculated. E Western blot assay was used to detect the protein expression of GPX4 and USP10 in subcutaneous xenografts derived from A549 cells. F , G Immunohistochemistry and immunofluorescence assays were used to detect the expression of GPX4 and USP10 in xenografted tumors. Scale bar: 125 μm. Data are shown as mean ± SD, n = 5. *** p < 0.01, * p < 0.001

    Article Snippet: The membrane was incubated overnight at 4 °C with primary antibodies against GPX4 (Abmart, T56959F), USP10 (SAB, 49,700), SLC7A11 (ABclonal, A2413), SLC3A2 (ABclonal, A3658), ACSL4 (Wanleibio, WL06242), and GAPDH (ZSGB-BIO, TA-08, AB_2747414).

    Techniques: In Vivo, Injection, Transfection, Western Blot, Expressing, Derivative Assay, Immunohistochemistry, Immunofluorescence

    Chronic stress-pre-incubated cells fail to disassemble polysomes by acute stress U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 1 μM Tg (thapsigargin), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (A) Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, G3BP1, G3BP2, caprin1, USP10, and β-actin. Three blots were taken for each of the three biological replicates (independent experiments). The same colors of the circle dot in the graph are from the same independent experiment. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗ <0.05, p ∗∗ <0.01, p ∗∗∗ <0.001, p ∗∗∗∗ <0.0001). (B–F) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; (B) 24 h pre-incubation of SA, blue; 1 h SA with 24 h pre-incubation of SA, green. (C) 24 h pre-incubation of Tg, blue; 1 h SA with 24 h pre-incubation of Tg, green. (D) 24 h pre-incubation of CCCP, blue; 1 h SA with 24 h pre-incubation of CCCP; green. (E) 24 h pre-incubation of HBSS, blue; 1 h SA with 24 h pre-incubation of HBSS; green. (F) The polysome/monosome ratio was calculated with normalization by each of NT as a control.

    Journal: iScience

    Article Title: Chronic stress antagonizes formation of stress granules

    doi: 10.1016/j.isci.2025.114556

    Figure Lengend Snippet: Chronic stress-pre-incubated cells fail to disassemble polysomes by acute stress U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 1 μM Tg (thapsigargin), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (A) Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, G3BP1, G3BP2, caprin1, USP10, and β-actin. Three blots were taken for each of the three biological replicates (independent experiments). The same colors of the circle dot in the graph are from the same independent experiment. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗ <0.05, p ∗∗ <0.01, p ∗∗∗ <0.001, p ∗∗∗∗ <0.0001). (B–F) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; (B) 24 h pre-incubation of SA, blue; 1 h SA with 24 h pre-incubation of SA, green. (C) 24 h pre-incubation of Tg, blue; 1 h SA with 24 h pre-incubation of Tg, green. (D) 24 h pre-incubation of CCCP, blue; 1 h SA with 24 h pre-incubation of CCCP; green. (E) 24 h pre-incubation of HBSS, blue; 1 h SA with 24 h pre-incubation of HBSS; green. (F) The polysome/monosome ratio was calculated with normalization by each of NT as a control.

    Article Snippet: Rabbit Polyclonal USP10 antibody , Bethyl Laboratories Inc. , A300-900A; RRID: AB_625312.

    Techniques: Incubation, Control, Western Blot