human axin1  (OriGene)

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a Pan-cancer analysis of TRIM15 expression. mRNA expression levels (log2(TPM + 1)) of TRIM15 across different tumor types from The Cancer Genome Atlas (TCGA) were shown as box plots, with the midline representing the mean value. b HEK293T cells transfected with the indicated plasmids were subjected to Co-IP. IP samples and whole cell lysates (WCL) were analyzed by Western blot. c Co-IP analysis of the interaction between Flag-TRIM15 and endogenous Axin1 in HCT116 cells. IP samples and WCL were analyzed by Western blot. d Cell lysates derived from HCT116 cells were analyzed by co-IP with control IgG or anti-Axin1 antibody. IP samples and WCL were analyzed by Western blot. e Flag-Axin1, purified from HEK293T cells, was incubated with either immobilized GST or GST-TRIM15, both of which were purified from bacteria. The pulldown samples and input were analyzed by SDS–PAGE followed by Western blot and/or Ponceau S staining. f Immunofluorescence staining of Flag-TRIM15 and GFP-Axin1 in HeLa cells. Nuclei (blue) were counterstained with DAPI. Scale bar represents 20 μm. g Schematic illustration of full-length and truncated Axin1. h Flag-TRIM15, purified from HEK293T cells, was incubated with immobilized GFP-fused Axin1 or its fragments, all of which were also purified from HEK293T cells. The pulldown samples and input were analyzed by SDS–PAGE followed by Western blot. i HEK293T cells transfected with TRIM15-YFP along with either Flag-Axin1 or Flag-Axin1 ΔD plasmids were subjected to Co-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot.

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Control, Purification, Incubation, Bacteria, SDS Page, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a HEK293T cells transfected with the indicated plasmids were subjected to Co-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot. b Flag-Axin1, purified from HEK293T cells, was incubated with either immobilized GST or GST-fused TRIM15 proteins, all of which were purified from bacteria. The pulldown samples and input were analyzed by Western blot and/or Ponceau S staining. c Purified Flag-TRIM15 or Flag-TRIM15 ΔRING proteins were incubated with immobilized GFP-fused Axin1 or E fragment. The pulldown and input samples were analyzed by Western blot. d Immunofluorescence staining of Flag-TRIM15 ΔRING and GFP-Axin1 in HeLa cells. Nuclei (blue) were counterstained with DAPI. Scale bar represents 20 μm. e Purified Flag-TRIM15 or its deletion proteins were incubated with immobilized GFP-Axin1. The pulldown samples and input were analyzed by Western blot. f Purified Flag-Axin1 was incubated with immobilized GST, GST-fused TRIM15, or its deletion proteins. The pulldown and input samples were analyzed by Western blot and/or Ponceau S staining. g Cell lysates from HEK293T cells transfected with the indicated plasmids were boiled in SDS-containing buffer, diluted, and d-IP using anti-Flag beads. IP samples and WCL were analyzed by Western blot. h HEK293T cells transfected with Flag-Axin1 with or without GFP-TRIM15. Protein stability was examined by collecting samples at the indicated time after CHX (100 μg/mL) treatment.

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Purification, Incubation, Bacteria, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: SPR (BIAcore) analysis of the dissociation constant ( K d ) for Axin1 ( a ) or TRIM15 ( b ) from immobilized Axin1. Axin1 and Axin1 interaction had a calculated K d of 147 nM, Axin1 and TRIM15 interaction had a calculated K d of 37.8 nM. c HCT116 cells were transfected with control siRNA (siCtrl) or TRIM15 siRNA (siTRIM15), followed by a sucrose density gradient centrifugation experiment to assess the polymerization degree of Axin1 protein. Immunofluorescence staining of Axin1 in DLD-1 cells transfected with siCtrl or TRIM15 siTRIM15 for 48 h. Representative confocal images of endogenous Axin1 ( d ) and the number of Axin1 polymers per cell ( e ). Data are Mean ± SD. Scale bar, 20 μm. f HEK293T cells transfected with the indicated plasmids were subjected to Co-IP. IP and WCL samples were examined by Western blot. HCT116 cells were transfected with siCtrl and siTRIM15 for 72 h, followed by Co-IP using IgG or anti-Axin1 ( g ) or anti-β-TrCP ( h ) antibodies. IP and WCL were analyzed by Western blot. Cells were treated with MG132 (20 μM) for 6 h before lysis. i An in vitro kinase assay was performed by incubation of GST-β-catenin with the indicated proteins purified from HEK293T cells. The samples were analyzed by Western blot. GST-β-catenin was purified from bacteria and shown by Coomassie blue staining.

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Transfection, Control, Gradient Centrifugation, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Lysis, In Vitro, Kinase Assay, Incubation, Purification, Bacteria

Journal: Cell Death & Disease

Article Title: Targeting TRIM15-mediated Axin1 depolymerization suppresses Wnt signaling and inhibits colorectal cancer growth

doi: 10.1038/s41419-025-08400-7

Figure Lengend Snippet: a Immunoblotting of total cell lysates from HCT116 wild type and TRIM15 KO cells treated with DMSO or IWR-1 (50 μM) for 48 h. b AlphaFold prediction of the complex formed by the Axin1 D domain and TRIM15 CC domain. P9715 included an HIV TAT peptide (red) and a peptide derived from the Axin1 D domain. c SPR (BIAcore) analysis of the dissociation constant ( K d ) for P9715 from immobilized TRIM15. The interaction between P9715 and TRIM15 had a calculated K d of 59.2 μM. d HEK293T cells were transfected with the indicated plasmids and then subjected to Co-IP with anti-Flag beads. Cells were treated with P9715 (50 and 100 μM) for 36 h before cell lysis. e HCT116 cells were treated with P9715 (100 μM) for 48 h, followed by the sucrose density gradient centrifugation experiment to assess the polymerization degree of Axin1 protein. f Immunoblotting of total cell lysates derived from HCT116 cells treated with IWR-1 (50 μM), P9715 (100 μM), or their combination for 72 h. Xenograft tumor formation by HCT116 cells in nude mice that were treated with IWR-1, P9715, or their combination. The tumor volume over time ( g ) tumor images ( h ) and tumor weight ( n = 5 animals) ( i ) were shown. j The proposed model. Data are Mean ± SD. One-way ANOVA for ( g , i ).

Article Snippet: Antibody against Axin1 (AF-3287-SP) was purchased from R&D Systems.

Techniques: Western Blot, Derivative Assay, Transfection, Co-Immunoprecipitation Assay, Lysis, Gradient Centrifugation

Journal: PLoS ONE

Article Title: Human Sarcoma Growth Is Sensitive to Small-Molecule Mediated AXIN Stabilization

doi: 10.1371/journal.pone.0097847

Figure Lengend Snippet: ( A ) U2OS cells infected with LVTCF-Luciferase and LVTA-Renilla were exposed to different amounts of SEN461 and the Wnt transcriptional activity was measured 24 h later. ( B ) The effect on Wnt transcriptional activity after inducible (10 ng/ml of doxycyclin) lentiviral infection with TCF4dn was measured by reporter activity. ( C ) Representative images for U2OS cells expressing Axin1-GFP (green) stained for P-β-catenin S33/S37/T41 (red) and their co-localization (yellow). Scale bar = 10 µm. U2OS cells, transiently transfected with GFP-tagged AXIN1 and stimulated with Wnt3a CM were either treated overnight with 10 µmol/L of SEN461 or with the inactive analogue SEN973 at the same concentration for the same length of time. ( D ) The bar-graph showed the result of three independent experiments where the number of Axin1/P-β-catenin co-localization puncta was counted in 5 different fields and normalized vs. untreated-Wnt3a-CM control. Data represent means ± SEM. ***, P<0.05 relative to control cells (Student t test). ( E ) Quantitative RT-PCR assay to measure the effect of SEN461 treatment after Wnt3a CM stimulation on the mRNA level of the Wnt target gene AXIN2 . Data represent means ± SEM. ***, P<0.0001 relative to Wnt3a stimulated cells (Student t test).

Article Snippet: Human AXIN1 and WNT3A cDNAs were purchased from Origene as “transfection ready” plasmids.

Techniques: Infection, Luciferase, Activity Assay, Expressing, Staining, Transfection, Concentration Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Human Sarcoma Growth Is Sensitive to Small-Molecule Mediated AXIN Stabilization

doi: 10.1371/journal.pone.0097847

Figure Lengend Snippet: ( A ) Summary of the half-maximal inhibitory concentration (IC 50 ) for the four sarcoma lines examined in soft agar is shown, and ranked from lowest to highest. ( B, C ) IC 50 for U2OS and HT-1080 cancer cells after XAV939 and SEN973 is shown, determined from the soft agar assay. Soft agar data (from two independent experiments) represent means ± SEM. ( D, E ) Western blotting analysis of Axin1 and β-catenin levels after SEN461 treatment in 143B and G292 osteosarcoma cells.

Article Snippet: Human AXIN1 and WNT3A cDNAs were purchased from Origene as “transfection ready” plasmids.

Techniques: Concentration Assay, Soft Agar Assay, Western Blot

Journal: PLoS ONE

Article Title: Human Sarcoma Growth Is Sensitive to Small-Molecule Mediated AXIN Stabilization

doi: 10.1371/journal.pone.0097847

Figure Lengend Snippet: ( A, D ) Effect of SEN461 or XAV939 on Wnt target genes mRNA expression measured by quantitative RT-PCR in U2OS and HT-1080 cells after 10 µmol/L treatment. Data (collected from three independent experiments) represent means ± SEM. **, P<0.05 ***, P<0.005 relative to control cells (Student t test). ( B, E ) Western blotting analysis of U2OS and HT-1080 cell lines treated with SEN461 or XAV939 overnight. Cytoplasmic cell lysates were probed with anti-Axin1, anti-TNKS1/2 and anti-Tubulin as loading control. ( C, F, G ) Western blotting analysis of U2OS and HT-1080 treated with SEN461 or XAV939 overnight. Cytoplasmic and nuclear cell lysates were then probed with anti-c-Myc, anti-p21 and anti-β-actin as loading control. The asterisk represents a background band migrating below the p21 band.

Article Snippet: Human AXIN1 and WNT3A cDNAs were purchased from Origene as “transfection ready” plasmids.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Human Sarcoma Growth Is Sensitive to Small-Molecule Mediated AXIN Stabilization

doi: 10.1371/journal.pone.0097847

Figure Lengend Snippet: ( A ) U2OS and HT-1080 cells were transiently transfected with AXIN1 cDNA. Cell lysates were then analyzed by Western blotting and probed with anti-Axin1, anti-c-Myc and anti-β-actin as loading control. ( B, C ) Ectopic expression of AXIN1 in U2OS and HT-1080 cells produced a strong reduction in their ability to grow in anchorage-independent fashion. Soft agar data (from two independent experiments) represent means ± SEM. *, P<0.05 ***, P<0.0005 relative to control cells (Student t test).

Article Snippet: Human AXIN1 and WNT3A cDNAs were purchased from Origene as “transfection ready” plasmids.

Techniques: Transfection, Western Blot, Expressing, Produced

Journal: Molecules and cells

Article Title: BK Channel Deficiency in Osteoblasts Reduces Bone Formation via the Wnt/β-Catenin Pathway.

doi: 10.14348/molcells.2021.0004

Figure Lengend Snippet: Fig. 5. Kcnma1 regulates β-catenin expression via canonical Wnt signaling. (A and B) Protein expression and quantitative analysis of β-catenin, Axin1 and USP7 in bone tissues from Osterix-Cre(–); Kcnma1f/f and Osterix-Cre(+); Kcnma1f/f mice. (C and D) western blot analysis of β-catenin, Runx2 and phosphorylated levels of GSK3β in osteoblasts isolated from the indicated mice. (E and F) Protein expression levels and quantitative analysis by western blot of Axin1 and USP7 in osteoblasts. Data are presented as mean ± SD. *P < 0.05, **P < 0.01.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk solution at room temperature and incubated at 4°C overnight with primary antibodies against BK (ab3586; Abcam, UK), β-catenin (51067-2-AP; Proteintech, USA), Runt-related transcription factor 2 (Runx2) (ab76956; Abcam), Col1a1 (A1352; Abclonal, USA), GSK3β (12456T; Cell Signaling Technology, USA), p-GSK3β (67558-1-Ig; Proteintech), USP7 (66514-1-Ig; Proteintech), and Axin1 (AF3287; R&D Systems, USA).

Techniques: Expressing, Western Blot, Isolation

Journal: Molecules and cells

Article Title: BK Channel Deficiency in Osteoblasts Reduces Bone Formation via the Wnt/β-Catenin Pathway.

doi: 10.14348/molcells.2021.0004

Figure Lengend Snippet: Fig. 6. Schematic diagram of how Kcnma1 regulates bone formation through Wnt/β-catenin signaling. In the process of bone development, the BK channel plays an essential role in the activation of osteoblast proliferation and osteogenic differentiation. When BK channels are unable to function normally, they stunt bone growth by inhibiting cytosol-nucleus transportation of β-catenin transportation in the canonical Wnt signaling pathway. USP7 maintains the stability of Axin1 and facilitates the degradation of β-catenin in osteoblasts, which decreases the expression of the transcription factor Runx2 and results in bone disorders.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk solution at room temperature and incubated at 4°C overnight with primary antibodies against BK (ab3586; Abcam, UK), β-catenin (51067-2-AP; Proteintech, USA), Runt-related transcription factor 2 (Runx2) (ab76956; Abcam), Col1a1 (A1352; Abclonal, USA), GSK3β (12456T; Cell Signaling Technology, USA), p-GSK3β (67558-1-Ig; Proteintech), USP7 (66514-1-Ig; Proteintech), and Axin1 (AF3287; R&D Systems, USA).

Techniques: Activation Assay, Expressing