wdr5 rabbit pab  (Bethyl)

Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) MYC transcript levels in control and MEN1-KO cells as determined by RT-qPCR. ns p > 0.05, * p ≤ 0.05. ( B ) MYC transcript counts from RNA-seq of A673 control and MEN1-KO tumors. ( C ) Western blot of MYC and Vinculin levels in A673 and TC32 control and MEN1-KO cells ( D ) Western blot of MYC and Vinculin in tumors derived from A673 control and MEN1-KO cells. ( E ) Co-immunoprecipitations with an anti-Menin antibody were performed on A673, CHLA10 and U2OS nuclear extracts and immunoblotted for Menin, MYC, MLL2 and WDR5. ( F ) Co-immunoprecipitations with an anti-MYC antibody were performed on A673 and CHLA10 nuclear extracts and immunoblotted for MYC, Menin, MAX, MLL2 and WDR5.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: Control, Quantitative RT-PCR, RNA Sequencing, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

doi: 10.1101/2025.11.10.687648

Figure Lengend Snippet: ( A ) Incucyte proliferation assay plotting percent confluence for A673 and TC32 cells treated with 0.1% DMSO or 10 µM VTP50469 starting at time 0. Representative of n=3-4). ( B and C ) Invasion of cells from a sphere of A673 cells embedded in rat tail collagen and treated with 0.1% DMSO or 10 µM VTP50469 for 5 days. ( B ) Representative phase and phalloidin (red)/DAPI (blue) stained images are shown (scale bars=100 µm). ( C ) Plot of circularity quantified from replicate spheroids treated as in ( B ). ( D ) A673 and TC32 cells were treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours and genes that were significantly downregulated in VTP50469-treated cells (padj<0.05) were overlapped with genes downregulated in MEN1-KO cells. The top 5 enriched Hallmark pathways for the overlapping downregulated genes in each cell line are shown along with the overlap/total genes for each pathway. The green arrows indicate pathways that were reactivated in A673 MEN1-KO tumors (see ). ( E ) MYC transcript levels in A673, CHLA10 and TC32 cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours as determined by RT-qPCR (ns p > 0.05). ( F ) Western of MYC and Vinculin in cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours. ( G ) Co-immunoprecipitations with an anti-Menin antibody were performed on nuclear extracts from A673 and CHLA10 cells treated with 0.1% DMSO or 10 µM VTP50469 for 72 hours and immunoblotted for Menin, MYC, MLL2 and WDR5.

Article Snippet: Membranes were blocked with Intercept (TBS) Blocking Buffer (LICORbio 927-60001) and probed for the primary antibodies GAPDH Rabbit mAb (Cell Signaling 2118), MAX Rabbit pAb (Cell Signaling 4739), Menin Goat pAb (Bethyl A300-106A), MLL1-C (C-terminal) Rabbit pAb (Bethyl A300-374A), MLL2-C (C-terminal) Rabbit mAb (Cell Signaling 63735), MYC Rabbit mAb (Cell Signaling 18583), Vinculin Rabbit mAb (Cell Signaling 13901) or WDR5 Rabbit pAb (Bethyl A302-430A) followed by the secondary antibody Goat anti-Rabbit 800CW (Licor 926-32211), Donkey anti-Rabbit 800CW (Licor 926-32213) or Donkey anti-Goat 680RD (Licor 926-68074).

Techniques: Proliferation Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Development and Characterization of Triazole-Based WDR5 Inhibitors for the Treatment of Glioblastoma

doi: 10.1101/2025.07.29.667410

Figure Lengend Snippet: (A) Structures and potencies of previously reported WDR5 WIN-site inhibitors. (B) Structures of all novel WIN-site inhibitors profiled herein. (C) Representative data for the biochemical TR-FRET assay measuring compound-dependent displacement of a labelled WIN-site peptide from human WDR5. Representative data from a single replicate performed in technical quadruplicate are presented as mean +/- SD. (D) Overlay of WDR5: C16 (green; PDB: 6UCS) and WDR5: C16-TZ (cyan, PDB: 9NCW) co-crystal structures reveals a conserved binding mode at the WIN-site, including especially a bidentate J-bond to Cys261.

Article Snippet: Gels were transferred to PVDF with a Trans-Blot Turbo (Biorad), probed with primary antibodies to WDR5 (1:1000, rb CST #13105) and ACTB (1:3,000 ms #CST3700), and amplified with 1:5,000 LI-COR goat secondary antibodies (IRDYE680RD Anti-Mouse & IRDYE800CW Anti-Rabbit).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Development and Characterization of Triazole-Based WDR5 Inhibitors for the Treatment of Glioblastoma

doi: 10.1101/2025.07.29.667410

Figure Lengend Snippet: (A) Melting curve for WDR5 (green) and ACTB (red) determined by CETSA-WB following a two hour treatment of L0 CSCs with either DMSO (top) or 10 μM C16 (bottom). (B) Isothermal CETSA-WB (70°C) for L0 CSCs treated for two hours with varying doses of the potent binder C3TD078 (top) or the weak binder C3TD343 (bottom). (C) Correlation between biochemical potency (TR-FRET K i ) and cellular potency (L0 CETSA K d ) as determined for n = 17 different WDR5 inhibitors. (D) Washout experiment following the treatment of L0 CSCs for two hours with either DMSO (top) or 5 μM C3TD078 (bottom) followed by compound washout. Samples were prepared for isothermal CETSA-WB at the indicated timepoints post-washout. (E) RT-qPCR analysis for the indicated genes after treating L0 CSCs with the indicated doses of the potent inhibitor C3TD078 (left) or the weak inhibitor C3TD424 (right) for 72 hours. Data are presented as mean +/- SD from a single biological replicate completed in technical triplicate. (F) Same as in (E) but with DI318 CSCs.

Article Snippet: Gels were transferred to PVDF with a Trans-Blot Turbo (Biorad), probed with primary antibodies to WDR5 (1:1000, rb CST #13105) and ACTB (1:3,000 ms #CST3700), and amplified with 1:5,000 LI-COR goat secondary antibodies (IRDYE680RD Anti-Mouse & IRDYE800CW Anti-Rabbit).

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Development and Characterization of Triazole-Based WDR5 Inhibitors for the Treatment of Glioblastoma

doi: 10.1101/2025.07.29.667410

Figure Lengend Snippet: Synthesis of triazole-based WDR5 inhibitor C16-TZ .

Article Snippet: Gels were transferred to PVDF with a Trans-Blot Turbo (Biorad), probed with primary antibodies to WDR5 (1:1000, rb CST #13105) and ACTB (1:3,000 ms #CST3700), and amplified with 1:5,000 LI-COR goat secondary antibodies (IRDYE680RD Anti-Mouse & IRDYE800CW Anti-Rabbit).

Techniques: