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Cell Signaling Technology Inc α flag
α Flag, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/%CE%B1+wdr5/us12545649-951-21-26?v=Cell+Signaling+Technology+Inc
Average 95 stars, based on 97 article reviews

α flag - by Bioz Stars, 2026-07

95/100 stars

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Journal: Cell reports

Article Title: MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

doi: 10.1016/j.celrep.2023.113120

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used are α-MATR3 (1:200, Thermo Fisher Scientific), α-DUX4 (1:50, P2B1, Sigma-Aldrich) and α-WDR5 (1:200, Cell Signaling Technology).

Techniques: Virus, Recombinant, Electron Microscopy, Luminescence Assay, In Situ, Mutagenesis, Transfection, SYBR Green Assay, Plasmid Preparation, Software

WDR5 is upregulated by FBXW7 depletion and interacts with FBXW7 independently of KMT2D. A , HeLa were transfected twice with siRNA against Gl2 or FBXW7 and harvested 72 h after the first transfection. ∗ p < 0.05, unpaired two-way Student’s t test with Welch’s correction, n = 3. B , HeLa were transfected twice with siRNA against Gl2 or FBXW7 and harvested 72 h after the first transfection. ns p > 0.05, unpaired two-way Student’s t test with Welch’s correction, n = 3. C , HCT116 WT and FBXW7 KO cell lines were transfected twice with siRNA against Gl2 or KMT2D and harvested 72 h after the first transfection. ∗∗∗ p < 0.001, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3. D , GFP-WDR5 WT or a F133A mutant were overexpressed in HEK293T cells for 24 h and immunoprecipitated using GFP-trap beads. ns p > 0.05, unpaired two-way Student’s t test with Welch’s correction, n =3. E , endogenous WDR5 or FBXW7 were immunoprecipitated from HEK293T lysates using specific antibodies and protein A or protein G Sepharose. F , purified recombinant GST-WDR5 and His-FBXW7 were combined, and GST pulldowns were performed using reduced glutathione on CL-4B beads. GST, glutathione- S -transferase; HEK293T, human embryonic kidney 293T cell line; mIgG, mouse control immunoglobulin G; ns, not significant; rIgG, rabbit control immunoglobulin G.

Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: WDR5 is upregulated by FBXW7 depletion and interacts with FBXW7 independently of KMT2D. A , HeLa were transfected twice with siRNA against Gl2 or FBXW7 and harvested 72 h after the first transfection. ∗ p < 0.05, unpaired two-way Student’s t test with Welch’s correction, n = 3. B , HeLa were transfected twice with siRNA against Gl2 or FBXW7 and harvested 72 h after the first transfection. ns p > 0.05, unpaired two-way Student’s t test with Welch’s correction, n = 3. C , HCT116 WT and FBXW7 KO cell lines were transfected twice with siRNA against Gl2 or KMT2D and harvested 72 h after the first transfection. ∗∗∗ p < 0.001, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3. D , GFP-WDR5 WT or a F133A mutant were overexpressed in HEK293T cells for 24 h and immunoprecipitated using GFP-trap beads. ns p > 0.05, unpaired two-way Student’s t test with Welch’s correction, n =3. E , endogenous WDR5 or FBXW7 were immunoprecipitated from HEK293T lysates using specific antibodies and protein A or protein G Sepharose. F , purified recombinant GST-WDR5 and His-FBXW7 were combined, and GST pulldowns were performed using reduced glutathione on CL-4B beads. GST, glutathione- S -transferase; HEK293T, human embryonic kidney 293T cell line; mIgG, mouse control immunoglobulin G; ns, not significant; rIgG, rabbit control immunoglobulin G.

Article Snippet: Proteins were detected using following antibodies: α-FBXW7α (catalog no.: A301-720), α-Rbbp5 (catalog no.: A300-109A), α-KMT2D (catalog no.: A300-BL1185), α-MLL1 (catalog no.: A300-374A), and α-SETD1A (catalog no.: A300-289A) antibodies were obtained from Bethyl. α-WDR5 (G9), α-cyclin E1 (HE12), and α-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology, Inc; α-FLAG-tag (F3165), α-vinculin (V9131), and α-tubulin (T6074) antibodies were purchased from Sigma. α-c-Myc (9402) antibody was purchased from Cell Signaling Technology; α-HA-tag (16B12) antibody was purchased from Babco; and the α-GFP antibody was produced in-house.

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Purification, Recombinant, Control

WDR5 protein stability is regulated by FBXW7-mediated ubiquitination. A , FLAG-WDR5 was overexpressed in DLD1 WT and FBXW7 KO cell lines for 24 h, followed by blockage of protein synthesis with 300 μg/ml cycloheximide (CHX) for the indicated durations prior to harvest. Protein half-lives were determined by fitting to a one-phase decay model, n = 3. + marks a nonspecific band. B , DLD1 WT and FBXW7 KO cell lines were treated with 300 μg/ml CHX for the indicated durations prior to harvest. C , FLAG-WDR5 was overexpressed in DLD1 WT for 24 h, followed by inhibition of protein synthesis with 300 μg/ml CHX in the presence of 10 μM MG132 or DMSO for the indicated durations prior to harvest. Normalized protein levels were fit to a one-phase decay model to determine protein half-lives, n = 3. D , FLAG-FBXW7 or FLAG-FBXW7 ΔF-box (amino acids 284–321 deletion) were overexpressed in HEK293T for 48 h, followed by inhibition of protein synthesis with 100 μg/ml CHX for the indicated durations prior to harvest. Normalized protein levels were fit to a one-phase decay model to determine protein half-lives, n = 3. E , the indicated proteins were overexpressed in HEK293T for 24 h, and the 26S proteasome was blocked by 10 μM MG132 for 5 h prior to harvest. FLAG-WDR5 was immunoprecipitated using α-FLAG M2 beads in the presence of 20 mM N -ethylmaleimide. DMSO, dimethyl sulfoxide; HEK293T, human embryonic kidney 293T cell line; l.e., long exposure; s.e., short exposure.

Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: WDR5 protein stability is regulated by FBXW7-mediated ubiquitination. A , FLAG-WDR5 was overexpressed in DLD1 WT and FBXW7 KO cell lines for 24 h, followed by blockage of protein synthesis with 300 μg/ml cycloheximide (CHX) for the indicated durations prior to harvest. Protein half-lives were determined by fitting to a one-phase decay model, n = 3. + marks a nonspecific band. B , DLD1 WT and FBXW7 KO cell lines were treated with 300 μg/ml CHX for the indicated durations prior to harvest. C , FLAG-WDR5 was overexpressed in DLD1 WT for 24 h, followed by inhibition of protein synthesis with 300 μg/ml CHX in the presence of 10 μM MG132 or DMSO for the indicated durations prior to harvest. Normalized protein levels were fit to a one-phase decay model to determine protein half-lives, n = 3. D , FLAG-FBXW7 or FLAG-FBXW7 ΔF-box (amino acids 284–321 deletion) were overexpressed in HEK293T for 48 h, followed by inhibition of protein synthesis with 100 μg/ml CHX for the indicated durations prior to harvest. Normalized protein levels were fit to a one-phase decay model to determine protein half-lives, n = 3. E , the indicated proteins were overexpressed in HEK293T for 24 h, and the 26S proteasome was blocked by 10 μM MG132 for 5 h prior to harvest. FLAG-WDR5 was immunoprecipitated using α-FLAG M2 beads in the presence of 20 mM N -ethylmaleimide. DMSO, dimethyl sulfoxide; HEK293T, human embryonic kidney 293T cell line; l.e., long exposure; s.e., short exposure.

Techniques: Ubiquitin Proteomics, Inhibition, Immunoprecipitation

The regulation of WDR5 by FBXW7 depends in part on GSK3β. A , FLAG-FBXW7 was overexpressed in HEK293T for 24 h, and all cultures were treated with 5 μM MLN4924 for 5 h prior to harvest. FLAG-FBXW7 was immunoprecipitated, and the indicated sample was dephosphorylated with λ-phosphatase at 30 °C for 3 h. B , GFP-WDR5 was overexpressed in HEK293T for 24 h, and cultures were treated with 5 μM CHIR-99021 or DMSO for 5 h prior to cell harvest, as indicated. GFP-WDR5 was immunoprecipitated using GFP-trap beads. ∗∗ p < 0.01, unpaired two-way Student’s t test with Welch’s correction, n = 3. C , U2OS cells were transfected twice with 30 nM against GL2 or GSK3β and harvested 72 h after the first transfection. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA, n = 3. D , GFP-WDR5 and Myc-GSK3β were overexpressed in HEK293T cells for 24 h. GFP-WDR5 was immunoprecipitated using GFP-trap beads. E , the indicated proteins were overexpressed in HEK293T for 24 h. 5 μM CHIR-99021 or 5 μM MLN4924 were added for 9 h and 10 μM MG132 for 5 h prior to harvest. FLAG-WDR5 was immunoprecipitated using α-FLAG M2 beads in the presence of 20 mM N -ethylmaleimide. F , FLAG-FBXW7 or FLAG-FBXW7 ARG (R465H, R479Q, and R505C mutations) were coexpressed with Myc-Cullin1 in HEK293T for 48 h and immobilized on α-FLAG M2 beads. In vitro ubiquitination assays of 200 nM GST-WDR5 or GST-c-Myc were performed with 170 nM Uba1, 250 nM of UbcH5b and CDC34, 30 μM ubiquitin, 200 nM His-GSK3β, 5 mM ATP, and immobilized FLAG-FBXW7 constructs at 37 °C for 90 min. DMSO, dimethyl sulfoxide; GSK3β, glycogen synthase kinase-3β; GST, glutathione- S -transferase; HEK293T, human embryonic kidney 293T cell line; l.e., long exposure; s.e., short exposure.

Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: The regulation of WDR5 by FBXW7 depends in part on GSK3β. A , FLAG-FBXW7 was overexpressed in HEK293T for 24 h, and all cultures were treated with 5 μM MLN4924 for 5 h prior to harvest. FLAG-FBXW7 was immunoprecipitated, and the indicated sample was dephosphorylated with λ-phosphatase at 30 °C for 3 h. B , GFP-WDR5 was overexpressed in HEK293T for 24 h, and cultures were treated with 5 μM CHIR-99021 or DMSO for 5 h prior to cell harvest, as indicated. GFP-WDR5 was immunoprecipitated using GFP-trap beads. ∗∗ p < 0.01, unpaired two-way Student’s t test with Welch’s correction, n = 3. C , U2OS cells were transfected twice with 30 nM against GL2 or GSK3β and harvested 72 h after the first transfection. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA, n = 3. D , GFP-WDR5 and Myc-GSK3β were overexpressed in HEK293T cells for 24 h. GFP-WDR5 was immunoprecipitated using GFP-trap beads. E , the indicated proteins were overexpressed in HEK293T for 24 h. 5 μM CHIR-99021 or 5 μM MLN4924 were added for 9 h and 10 μM MG132 for 5 h prior to harvest. FLAG-WDR5 was immunoprecipitated using α-FLAG M2 beads in the presence of 20 mM N -ethylmaleimide. F , FLAG-FBXW7 or FLAG-FBXW7 ARG (R465H, R479Q, and R505C mutations) were coexpressed with Myc-Cullin1 in HEK293T for 48 h and immobilized on α-FLAG M2 beads. In vitro ubiquitination assays of 200 nM GST-WDR5 or GST-c-Myc were performed with 170 nM Uba1, 250 nM of UbcH5b and CDC34, 30 μM ubiquitin, 200 nM His-GSK3β, 5 mM ATP, and immobilized FLAG-FBXW7 constructs at 37 °C for 90 min. DMSO, dimethyl sulfoxide; GSK3β, glycogen synthase kinase-3β; GST, glutathione- S -transferase; HEK293T, human embryonic kidney 293T cell line; l.e., long exposure; s.e., short exposure.

Techniques: Immunoprecipitation, Transfection, In Vitro, Ubiquitin Proteomics, Construct

WDR5 and cyclin E1 promote mitotic slippage. A , still images from live-cell imaging with U2OS T-Rex WDR5 showing different mitotic cell fates in response to 830 nM nocodazole. Arrows indicate cells undergoing mitotic arrest and slippage or apoptosis. B , U2OS T-Rex WDR5 were cultured in medium containing 2 μg/ml doxycycline for 4 days before the addition of 830 nM nocodazole and live-cell imaging. Mitotic cell fate of n = 50 cells was determined for each experiment. ∗ p < 0.05, two-way unpaired Student's t test, n = 3. C , U2OS Tet-Off cyclin E1 were cultured with or without 2 μg/ml doxycycline for 4 days. At days 2 and 3, cells were transfected with 30 nM siRNA targeting Gl2 of FBXW7, as indicated. About 48 h after the second transfection, 830 nM nocodazole were added and mitotic cell fates of n = 50 cells determined by live-cell imaging. ∗∗ p < 0.01, ∗ p < 0.05, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3. D , U2OS were transfected twice with 30 nM siRNA targeting Gl2, FBXW7, cyclin E1, or WDR5, as indicated. About 830 nM nocodazole were added 48 h after the second transfection, and mitotic cell fates of n = 50 cells were determined by live-cell imaging. ∗∗∗ p < 0.001, ∗∗ p < 0.01, p < 0.05, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3. ns, not significant.

Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: WDR5 and cyclin E1 promote mitotic slippage. A , still images from live-cell imaging with U2OS T-Rex WDR5 showing different mitotic cell fates in response to 830 nM nocodazole. Arrows indicate cells undergoing mitotic arrest and slippage or apoptosis. B , U2OS T-Rex WDR5 were cultured in medium containing 2 μg/ml doxycycline for 4 days before the addition of 830 nM nocodazole and live-cell imaging. Mitotic cell fate of n = 50 cells was determined for each experiment. ∗ p < 0.05, two-way unpaired Student's t test, n = 3. C , U2OS Tet-Off cyclin E1 were cultured with or without 2 μg/ml doxycycline for 4 days. At days 2 and 3, cells were transfected with 30 nM siRNA targeting Gl2 of FBXW7, as indicated. About 48 h after the second transfection, 830 nM nocodazole were added and mitotic cell fates of n = 50 cells determined by live-cell imaging. ∗∗ p < 0.01, ∗ p < 0.05, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3. D , U2OS were transfected twice with 30 nM siRNA targeting Gl2, FBXW7, cyclin E1, or WDR5, as indicated. About 830 nM nocodazole were added 48 h after the second transfection, and mitotic cell fates of n = 50 cells were determined by live-cell imaging. ∗∗∗ p < 0.001, ∗∗ p < 0.01, p < 0.05, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3. ns, not significant.

Techniques: Live Cell Imaging, Cell Culture, Transfection

$WDR5 and cyclin E1 are equally required for drug-induced polyploidy in response to antimicrotubule drugs. A , schematic depicting the protocol followed to determine drug-induced polyploidy of HCT116. HCT116 WT and FBXW7 KO were transfected twice with 30 nM siRNA and blocked in S-phase with 2 mM thymidine for 24 h. Cells were released into medium containing 500 nM taxol and harvested and fixed after 20 h. B , exemplary histograms showing DNA contents of HCT116 determined by FACS-Scan analysis. HCT116 were treated following the protocol in A , and DNA was stained with PI for 15 min at RT. Histograms are shown as modal counts and fraction of G2/M (4N) and polyploid (>4N) cells. C , HCT116 were transfected following the protocol from A but without synchronization and taxol treatment. Asynchronous cells were harvested 48 h after the second siRNA transfection. D , statistical analysis of B. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3 to 4. FACS, fluorescence-activated cell sorting; ns, not significant; PI, propidium iodide; RT, room temperature.$

Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: WDR5 and cyclin E1 are equally required for drug-induced polyploidy in response to antimicrotubule drugs. A , schematic depicting the protocol followed to determine drug-induced polyploidy of HCT116. HCT116 WT and FBXW7 KO were transfected twice with 30 nM siRNA and blocked in S-phase with 2 mM thymidine for 24 h. Cells were released into medium containing 500 nM taxol and harvested and fixed after 20 h. B , exemplary histograms showing DNA contents of HCT116 determined by FACS-Scan analysis. HCT116 were treated following the protocol in A , and DNA was stained with PI for 15 min at RT. Histograms are shown as modal counts and fraction of G2/M (4N) and polyploid (>4N) cells. C , HCT116 were transfected following the protocol from A but without synchronization and taxol treatment. Asynchronous cells were harvested 48 h after the second siRNA transfection. D , statistical analysis of B. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ns p > 0.05, one-way ANOVA with Tukey post hoc test, n = 3 to 4. FACS, fluorescence-activated cell sorting; ns, not significant; PI, propidium iodide; RT, room temperature.

Techniques: Transfection, Staining, Fluorescence, FACS

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