Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: USP20 directly interacts with GPX4. ( A ) Immunoblot analysis of target proteins in sorafenib-resistant 769-P and A549 cells following 24 h treatment with specific deubiquitinase inhibitors. ML-323: 10 μM, GSK2643943A: 10 μM, XL177A: 10 μM, Spautin-1: 10 μM, IU1: 10 μM, AZ1: 10 μM, MF-094: 10 μM, LDN-57444: 10 μM, TCID: 10 μM, PR-619: 10 μM, BAY11-7082: 10 μM, EOAI3402143: 2.5 μM, ML364: 10 μM, b-AP15: 0.5 μM. ( B ) HEK293T cells were transfected with GST-GPX4 and Flag-USP. The cell lysate was applied with GST pull-down, then analyzed with immunoblot for indicated proteins. ( C ) 769-P and A549 cells were subjected to immunoprecipitate with anti-GPX4 antibodies, then analyzed with immunoblot for indicated proteins. ( D ) Overview of USP20 structures. ( E-F ) HEK293T cells transfected with the indicated USP20 structures and Flag-GPX4 were subjected to pull-down with GSH beads or immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( G ) 769-P and A549 cells were subjected to subcellular fractionation, then analyzed with immunoblot for indicated proteins. ( H ) Overview of GPX4 isoforms structures. ( I ) HEK293T cells transfected with the indicated GPX4 isoforms and GST-USP20 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( J ) HEK293T cells transfected with the GST-USP20 WT/C154S and Flag-GPX4 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins.

Article Snippet: Primary antibodies against USP20 (cat. no. 17491-1-AP; in ), HA-Tag (cat. no. 51064-2-AP), FLAG-Tag (cat. no. 20543-1-AP), GST-Tag (cat. no. 66001-2-Ig), β-Actin (cat. no. 66009-1-Ig), His-Tag (cat. no. 66005-1-Ig), GPX4(cat.no.67763-1-Ig), ACSL4 (cat. no. 22401-1-AP), SLC7A11 (cat. no. 26864-1-AP), FSP1 (cat. no. 20886-1-AP), FTH (cat. no. 11682-1-AP), Vinculin (cat. no. 26520-1-AP), MEK (cat. no. 11049-1-AP), TOMM20 (cat. no. 11802-1-AP), and H3 (cat. no. 17168-1-AP) were purchased from ProteinTech.

Techniques: Western Blot, Transfection, Fractionation

Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: USP20 removes GPX4 K48-linked poly-ubiquitination. ( A ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, GST-USP20 and His-Ub. ( B ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, GST-USP20 WT/C154S and His-Ub. ( C ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, and His-Ub under the condition of the indicated concentration of GSK2643943A. ( D ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in USP20 knockdown HEK293T cells transfected with Flag-GPX4 and His-Ub. ( E ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, HA-USP20 and His-Ub WT or His-Ub K48−only . ( F ) Ubiquitination assay was used to analyze the ubiquitination of GPX4 in HEK293T cells transfected with Flag-GPX4, HA-USP20 and His-Ub WT or His-Ub K48R .

Article Snippet: Primary antibodies against USP20 (cat. no. 17491-1-AP; in ), HA-Tag (cat. no. 51064-2-AP), FLAG-Tag (cat. no. 20543-1-AP), GST-Tag (cat. no. 66001-2-Ig), β-Actin (cat. no. 66009-1-Ig), His-Tag (cat. no. 66005-1-Ig), GPX4(cat.no.67763-1-Ig), ACSL4 (cat. no. 22401-1-AP), SLC7A11 (cat. no. 26864-1-AP), FSP1 (cat. no. 20886-1-AP), FTH (cat. no. 11682-1-AP), Vinculin (cat. no. 26520-1-AP), MEK (cat. no. 11049-1-AP), TOMM20 (cat. no. 11802-1-AP), and H3 (cat. no. 17168-1-AP) were purchased from ProteinTech.

Techniques: Ubiquitin Proteomics, Transfection, Concentration Assay, Knockdown