trim21 flag  (Sino Biological)

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) GSEA plots showing pathway signatures that are enriched in MAP3K8 -high PDAC samples analyzed from the TCGA PDAC Firehose Legacy database. (B) Relative SRE reporter activity in HEK293T cells stably expressing EV or KRAS G12V and KRAS G12D -mutant HPAC and Pa01C cells treated with different concentrations of TPL2i for 48 h. Data were pooled from 2 to 3 independent experiments, each done in biological triplicates and presented as mean ± SEM (**** p < 0.0001, *** p < 0.0002, ** p < 0.0021 by two-way ANOVA). (C) IP western blots of HEK293T cells stably expressing EV or HA-TPL2 transfected with EV or FLAG-KRAS G12V . 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 protein in HEK293T cells stably expressing EV or FLAG-KRAS G12V treated with 10 μg/mL CHX for the indicated durations. (E) Representative fluorescence images and quantification showing in situ TPL2-TRIM4 punctate dots by Duolink PLA in HEK293T cells expressing EV or FLAG-KRAS G12V . Data are presented as mean ± SEM. **** p < 0.0001 by two-tailed t test, six fields per condition were analyzed. Scale bars, 50 μm). (F) Western blots and t 1/2 of endogenous TRIM4 protein in control or KRAS -silenced Pa01C cells treated with CHX (10 μg/mL) for the indicated durations. (G) Western blots and t 1/2 of endogenous TRIM4 protein in MIA Paca-2 cells pre-treated overnight with AMG-510 (0.2 μM), followed by (CHX 10 μg/mL) for the indicated durations. For (D), (F), and (G), the t 1/2 of the indicated proteins were calculated by comparing band intensities at t 0 and performing one-phase exponential decay analysis. Data from three independent experiments are quantified, with blots from one representative experiment shown.

Article Snippet: The pBabe expression FLAG-KRAD G12D was obtained from Dr. Adhikari (Washington University, St. Louis, MO, USA). pLVX puro TRIM21-GFP (Cat# 116941) and pLVX puro TRIM21 C54Y-GFP (Cat# 116942), pXL010-Wnt dual (GFP-Fire) reporter (Cat# 40588), pCMV-Pam-FL (Cat# 42570), pcDNA5FRT/TO-RNF185-GFP (Cat #113498), HA-GSK3β pcDNA3 (Cat #14753) were purchased from Addgene.

Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Western Blot, Transfection, Fluorescence, In Situ, Two Tailed Test, Control

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: The pBabe expression FLAG-KRAD G12D was obtained from Dr. Adhikari (Washington University, St. Louis, MO, USA). pLVX puro TRIM21-GFP (Cat# 116941) and pLVX puro TRIM21 C54Y-GFP (Cat# 116942), pXL010-Wnt dual (GFP-Fire) reporter (Cat# 40588), pCMV-Pam-FL (Cat# 42570), pcDNA5FRT/TO-RNF185-GFP (Cat #113498), HA-GSK3β pcDNA3 (Cat #14753) were purchased from Addgene.

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:

Article Snippet: The pBabe expression FLAG-KRAD G12D was obtained from Dr. Adhikari (Washington University, St. Louis, MO, USA). pLVX puro TRIM21-GFP (Cat# 116941) and pLVX puro TRIM21 C54Y-GFP (Cat# 116942), pXL010-Wnt dual (GFP-Fire) reporter (Cat# 40588), pCMV-Pam-FL (Cat# 42570), pcDNA5FRT/TO-RNF185-GFP (Cat #113498), HA-GSK3β pcDNA3 (Cat #14753) were purchased from Addgene.

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

Journal: Cell Death & Disease

Article Title: BCKDK regulates breast cancer cell adhesion and tumor metastasis by inhibiting TRIM21 ubiquitinate talin1

doi: 10.1038/s41419-023-05944-4

Figure Lengend Snippet: A The expression of talin1 was examined by western blot in HEK293T cells. B The expression of talin1 was examined by western blot in MDA-MB-231 cells. C Exogenous talin1 coimmunoprecipitated with TRIM21 in HEK293T cells. D The degradation protein level of talin1 detected in M231/Ctrl and M231/TRIM21 cells with CHX treatment (100 μg/mL) for indicated time intervals. E Co-IP shows that HA-ubiquitin is pulled down by talin1 in HEK293T cells. HEK293T cells were transfected with Myc-tagged talin1 and HA-ubiquitin, plus Flag-tagged TRIM21 or the vector controls. At 48 h post-transfection, cells were treated with MG132 for 4 h, and then lysates of these cells were subjected to IP with anti-Myc-conjugated beads. Immunoprecipitates were subjected to IB analysis for ubiquitinated talin1 with anti-ubiquitin or for indicated proteins. F HEK293T cells were transfected with Myc-tagged talin1 and HA-ubiquitin, plus Flag-tagged TRIM21, Flag-tagged BCKDK or their vector controls. At 48 h post-transfection, cells were lysed and subjected to IP with anti-Myc-conjugated beads. The immunoprecipitates were subjected to IB analysis for ubiquitinated talin1 with anti-ubiquitin or for indicated proteins. G IP analysis using anti-Myc antibody followed by IB analysis to detect HA-BCKDK and Flag-TRIM21 expression in HEK293T cells.

Article Snippet: A scrambled siRNA with a sequence lacking significant homology to the human genome database was used as the control siRNA (shMOCK). pCMV3-BCKDK-HA and pCMV3-TRIM21-Flag were purchased from Sino Biological. pCMV3-BCKDK-Flag was constructed by our laboratory. pReceiver-Lv202-TLN1-Myc was customized at GeneCopoeia.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation