gst tag  (Proteintech)

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

Journal: Materials Today Bio

Article Title: Sequential delivery of sinigrin and dabigatran by an in situ self-stabilizing dynamic hydrogel attenuates intervertebral disc degeneration

doi: 10.1016/j.mtbio.2026.102827

Figure Lengend Snippet: Metabolic regulation of nucleus pulposus vells by SIN and DAB through MAPK and NF-κB pathways. (A) Volcano plot analysis of DE genes (TNF-α vs. TNF-α+SIN). (B) KEGG enrichment analysis of DE genes (TNF-α vs. TNF-α+SIN). (C) GSEA indicated MAPK signalling pathway enrichment. (D) Heatmap of ten selected differentially expressed genes (TNF-α vs. TNF-α+SIN). (E) Western blot analysis of key proteins in the MAPK signalling pathway (p-ERK, p-JNK, p-p38, ERK, JNK, and p38) in the different treatment groups. (F) Quantitative analysis of the Western blot data for MAPK signalling pathway proteins. (G–H) Western blot and quantitative analysis of p-ERK, p-JNK, p-p38, ERK, JNK, and p38 treated with or without TNF-α in the absence or presence of SIN (10 μM) (PCDNA 3.1 vs. TNFR1-OE).(I–L) Integrated analysis of TNF-α vs. TNF-α+DAB DEGs: Volcano plot of DE genes (significance/fold change), KEGG pathway enrichment of DE genes, NF-κB signalling enrichment in GSEA, and heatmap of ten selected differentially expressed genes. (M) Western blot analysis of key proteins in the NF-κB signalling pathway (p-IκBα, p-p65, IκBα, and p65) in the different treatment groups. (N) Quantitative analysis of the Western blot data for the NF-κB signalling pathway proteins. (O–P) Western blot and quantitative analysis of p-p65 and p65 (PCDNA 3.1 vs. TNFR1-OE). (Q) Matched diagram of b and y ions. (R) Western blot analysis of RELA in the different treatment groups. (S) Western blot analysis confirmed the stabilization of p65 by DAB. (T) The affinity between DAB and the p65 target protein was estimated after incubation at different temperatures via Western blot analysis. (n = 3; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).

Article Snippet: The membranes were then incubated overnight with anti-GAPDH (1:50 000, AC054, abclone, China), anti-Col2α1(1:500, A19308, abclone, China), anti-Acan (1:1000, A8536, abclone, China), anti-SOX9 (1:1000, A19710, abclone, China), anti-MMP13 (1:1000, A1606, abclone, China), anti-ADAMTS4 (1:500, A2525, abclone, China), anti-iNOS (1:1000, 18985-1-AP, proteintech, China), anti-COX2 (1:1000, 12375-1-AP, proteintech, China), anti-ERK (1:2000, 11257-1-AP, proteintech, China), anti-Phospho-ERK (1:1000, 28733-1-AP, proteintech, China), anti-JNK (1:500, 51153-1-AP, proteintech, China), anti-Phospho-JNK (1:1000, 80024-1-RR, proteintech, China), anti-p38 (1:1000, A4771, abclone, China), anti-Phospho-p38 (1:500, AP1508, abclone, China), anti-p65 (1:5000, A19653, abclone, China), anti-Phospho-p65 (1:500, AP0475, abclone, China), anti-IκBα (1:500, A1187, abclone, China), anti-Phospho-IκBα (1:500, AP0420, abclone, China), anti-AMPK (1:5000, A28094, abclone, China), anti-Phospho-AMPK (1:500, AP0432, abclone, China), The membrane was incubated with HRP-conjugated Goat anti-rabbit IgG (H + L) (1:50 000, AS014, rabbit monoclonal, Abclone, China) and immunoreactive proteins were detected using enhanced chemiluminescence.

Techniques: Western Blot, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 directly interacts with DDX41 and regulates its phosphorylation. ( A ) HCECs transfected with either pcDNA3.1 (Flag) or pcDNA3.1 PIM1 (Flag-PIM1) were stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Cell lysates were immunoprecipitated using anti-Flag beads, and bound proteins were separated by SDS-PAGE and visualized by silver staining. ( B ) A GST pull-down assay was performed to examine direct binding between PIM1 and DDX41. ( C ) Endogenous interaction between PIM1 and DDX41 in HCECs was assessed by co-IP. HEK293T cells were cotransfected with Flag-PIM1 and Myc-DDX41 for 48 hours. (D) Co-IP was carried out with anti-Flag beads, and Myc-DDX41 was detected with anti-Myc antibody. ( E ) Reciprocal co-IP was performed using anti-Myc beads, and Flag-PIM1 was probed with anti-Flag antibody. ( F ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours. DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and DDX41 antibodies. ( G ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours, followed by DDX41 immunoprecipitation and immunoblotting with indicated antibodies. ( H ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Co-IP of DDX41 was performed, and associated proteins were analyzed by Western blot. ( I ) Quantification of protein levels in ( H ). ( J ) HCECs were pretreated with SGI-1776 (0 and 4 µM) for 12 hours prior to 12-hour stimulation with A. fumigatus hyphae. Co-IP assay was performed using anti-DDX41 antibody and immunoblotted with the antibodies indicated. ( K ) Quantification of protein levels in ( J ). ( L ) Recombinant His-PIM1 and GST-DDX41 were incubated in reaction buffer with or without phosphatase for 30 minutes. The protein levels of p-Ser, His-PIM1, and GST-DDX41 were detected by Western blot. Quantification of protein levels in F, G, and L is shown in A–C. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: Antibodies targeting PIM2 (25865-1-AP, 1:1000), PIM3 (33202-1-AP, 1:1000), β-actin (20536-1-AP, 1:50,000), GST (10000-0-AP, 1:2000), MYC (16286-1-AP, 1:2000), Flag (66008-4-Ig, 1:2000), His (66005-1-Ig, 1:2000), STING (19851-1-AP, 1:2000), IRF3 (11312-1-AP, 1:1000), and TBK1 (28397-1-AP, 1:1000) were sourced from Proteintech (Wuhan, China).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Western Blot, Recombinant, Incubation

Journal: Materials Today Bio

Article Title: A precise theranostic nanoplatform amplifies anti-tumor efficacy via copper ionophores and sonodynamic therapy

doi: 10.1016/j.mtbio.2026.102957

Figure Lengend Snippet: The anti-tumor immune mechanism of ICCP NPs. a) Schematic illustration of cuproptosis mechanism. b) Immunofluorescence analysis of DLAT and FDX1 in 4T1 cells following various treatments. c) Schematic diagram illustrating the synergistic induction of ICD by SDT and cuproptosis. d) Immunofluorescence analysis of CRT and HMGB1 in 4T1 cells following various treatments. e) Quantitative analysis of CRT fluorescence intensity. f) ATP content in 4T1 cells following various treatments.

Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit polyclonal antibodies against DLAT (13426-1-AP, 1:200), FDX1 (12592-1-AP, 1:250), CRT (10292-1-AP, 1:200), and HMGB1 (10829-1-AP, 1:200) (Proteintech, Wuhan, China).

Techniques: Immunofluorescence, Fluorescence

Journal: Materials Today Bio

Article Title: A precise theranostic nanoplatform amplifies anti-tumor efficacy via copper ionophores and sonodynamic therapy

doi: 10.1016/j.mtbio.2026.102957

Figure Lengend Snippet: ICCP NPs mediated anti-tumor immunity in vivo . a) Schematic diagram llustrating the synergistic anti-tumor immune mechanism of ICCP NPs through SDT and cuproptosis. b) Immunofluorescence analysis of CRT and HMGB1 across different treatments. c) Assessment of mature DC proportions (CD80 + /CD86 + ) in tumor tissues via flow cytometry and d) corresponding quantitative analysis. e) Assessment of CD4 + T cells proportions in tumor tissues via flow cytometry and f) corresponding quantitative analysis. g) Assessment of CD8 + T cells proportions in tumor tissues via flow cytometry and h) corresponding quantitative analysis.

Article Snippet: Cells were then incubated overnight at 4 °C with primary rabbit polyclonal antibodies against DLAT (13426-1-AP, 1:200), FDX1 (12592-1-AP, 1:250), CRT (10292-1-AP, 1:200), and HMGB1 (10829-1-AP, 1:200) (Proteintech, Wuhan, China).

Techniques: In Vivo, Immunofluorescence, Flow Cytometry