Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Schematic workflow of IP-mass spectrometry with HEK293T cells stably expressing FLAG-KRAS G12V or FLAG-RALA G23V . Selected protein hits with more than 10 significant sequence matches and enriched in KRAS G12V lysates by ≥3-fold are shown. An asterisk denotes known KRAS interactors based on BioGRID 4.4 . (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 transfected with EV (denoted as −), GFP-tagged WT, or enzymatically inactive (C54Y) TRIM21. 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots. (D) Western blots and t 1/2 of HA-TPL2 proteins in HEK293T cells stably expressing EV or TRIM21-GFP treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments is quantified with blots from one representative experiment shown. (E) Relative SRE-driven luciferase reporter activity in HEK293T cells transfected with EV or the indicated constructs. Data were pooled from three independent experiments, each done in triplicates, and are presented as mean ± SEM (****p < 0.0001). (F) Western blots and densitometric quantification of the indicated markers in Pa01C and HPAC cells stably expressing a scramble or two different shRNAs against TRIM21 . (G) GSEA plots showing KRAS and MEK oncogenic signatures in TRIM21 -silenced Pa01C cells.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Sequencing, Western Blot, Transfection, Luciferase, Activity Assay, Construct

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) IP-western blots of HEK293T cells stably expressing EV or FLAG-TRIM4 and transfected with EV or TRIM21-GFP. (B) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 cells transfected with a fixed amount of TRIM4 (6 μg in 10 cm 2 plates) and different amounts (1.5 μg, 3 μg, and 6 μg) of TRIM21. (C) Western blots and t 1/2 of endogenous TRIM4 protein in HEK293T cells transfected with the indicated cDNAs and treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments were quantified, with blots from one representative experiment shown. (D) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 and transfected with EV, TRIM21-GFP, and FLAG-KRAS G12V . (E) IP-western blots showing TRIM21 polyubiquitination in HEK293T cells transfected with EV, TRIM21-GFP, and/or HA-KRAS G12V . For (A), (B), (D), and (E), 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Volcano plot of KRAS G12V BioID experiments, which identified RNF185 and MYCBP2 as the two E3 ligases enriched by >2-fold through proximal biotinylation by BirA-KRAS G12V . Identification of the known KRAS G12V effectors BRAF and RALA confirmed the robustness of the data. EFR3A was confirmed and published previously. (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous RNF185 and TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or FLAG-tagged KRAS G12D treated with DMSO or MRTX1133 at 0.1 and 0.5 μM overnight and harvested for western blots. (D and E) Representative Duolink PLA fluorescence images and quantification of fluorescent puncta showing in situ interaction between (D) endogenous RNF185 and KRAS or (E) TRIM21-GFP and endogenous RNF185 in Pa01C and HPAC cells treated overnight with MRTX1133. Data presented as mean ± SEM (****p < 0.0001 by one-way ANOVA; six fields per condition were analyzed; scale bars, 20 μm). (F) IP-western blot of HEK293T cells stably expressing EV or HA-KRAS G12V and transiently transfected with TRIM21-GFP or/and FLAG-RNF185. (G and H) Western blots and t 1/2 of endogenous TRIM21 protein in (G) HEK293T FLAG-KRAS G12D cells stably expressing EV or GFP-RNF185 or (H) Pa01C cell stably expressing a scramble or shRNF185, following treatment with CHX (10 μg/mL) for the indicated durations. (I) Western blots and densitometric quantification of the indicated markers in scramble and two different RNF186 -silenced Pa01C cells. (J) Western blots and t 1/2 of endogenous TRIM21 and TRIM4 proteins in HPAC cells pre-treated overnight with DMSO or MRTX1133, followed by CHX (10 μg/mL) for the indicated durations. (K) Western blots and densitometric quantification showing the impact of overnight MRTX1133 treatment on the indicated markers in Pa01C and HPAC cells. For (G), (H), and (J), data from three independent experiments were quantified, with blots from one representative experiment shown.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Western Blot, Stable Transfection, Expressing, Fluorescence, In Situ, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Workflow of IP-mass spectrometry and enrichment plot demonstrating GSK3β as one of the top hits enriched in HEK293T cells expressing HA-TPL2 plus TRIM21-GFP when compared to HA-TPL2 alone. (B) IP-western blots confirming the interaction of HA-TPL2 with endogenous GSK3β when TRIM21 is overexpressed. (C and D) Bar graphs showing relative luciferase reporter activity in (C) 293T TOPFlash reporter cells transfected with EV or HA-TPL2 and (D) Pa01C and HPAC cells stably expressing a Wnt luciferase reporter treated overnight with TPL2i. (E) Bar graphs showing selected up- and downregulated Molecular Signatures Database (MSigDB) oncogenic signatures with normalized enrichment score and false discovery rate q values. Shown are GSEA plots focusing on Wnt and MEK signatures in MAP3K8 -silenced Pa01C cells. (F and G) Western blots and densitometric quantification of the indicated markers in (F) HEK293T cells stably expressing EV or HA-TPL2 and (G) Pa01C and HPAC cells treated overnight with TPL2i. (H) Relative Wnt luciferase reporter activity in Pa01C and HPAC cells treated with the indicated agents overnight. (I and J) Western blots and densitometric quantification of p-GSK3β and p-β-catenin in (I) Pa01C and HPAC cells stably expressing EV or TRIM21-GFP and (J) scramble or TRIM21 -silenced Pa01C cells. (K) GSEA plot showing the upregulated Wnt pathway signature in TRIM21 -silenced Pa01C cells. (L) IP-western blots showing GSK3β polyubiquitination in HEK293T cells stably expressing the indicated cDNAs. (M) Western blots and t 1/2 of endogenous GSK3β in Pa01C cells stably expressing EV or TRIM21-GFP treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments were quantified, with blots from one representative experiment shown. (N and O) Relative TOPFlash luciferase reporter activity in HEK293T cells stably expressing the indicated cDNAs (N) treated overnight with DMSO or GSK3β inhibitor and (O) treated overnight with TPL2i. (P) Relative Wnt luciferase reporter activity in Pa01C and HPAC cells treated overnight with the indicated agents. For (C), (D), (H), and (N)–(P), data were pooled from two or three independent experiments, each done in technical triplicates, and are presented as mean ± SEM. The p values were calculated from two-tailed t test or ANOVA for multiple groups. **** p < 0.0001, *** p < 0.0002, ** p < 0.0021, * p < 0.0332.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Mass Spectrometry, Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Stable Transfection, Two Tailed Test

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet:

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Ubiquitin Proteomics, Recombinant, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Ligation, Western Blot, Over Expression, Plasmid Preparation, Amplification, Clone Assay, Expressing, Software, Microscopy