Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 1 Co-immunoprecipitation of CtIP and AdE1A. (a) Lysates from 293 cells were immunoprecipitated with antibodies against Ad5E1A(M58) or CtBP1 (E12) as shown. Co-immunopre- cipitating CtIP was detected by western blotting. (b) Lysates from 911 and 293 cells were immunoprecipitated with two CtIP rabbit polyclonal antibodies. Immunoprecipitates were fractionated on ‘urea gels’ and co-immunoprecipitating Ad5E1A detected by western blotting. (c) Lysates from MCF7 cells infected (and mock infected) with Ad5wt were immunoprecipitated for Ad5E1A. Co- immunoprecipitating proteins were fractionated on SDS gels and CtIP identified by western blotting. (d) Lysates from MCF7 cells infected with dl520 or dl1135 were immunoprecipitated with rabbit antibodies against CtIP. Co-immunoprecipitating proteins were fractionated on ‘urea gels’ in the absence of SDS and western blotted for Ad5E1A. (e) Lysates from Ad12E1-transformed human and rat cells were immunoprecipitated with an antibody against Ad12E1A. Co-immunoprecipitating proteins were fractionated on gels run in the presence of SDS and blotted for CtIP. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein; SDS, sodium dodecylsulphate.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Immunoprecipitation, Western Blot, Infection, Transformation Assay, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 2 CtIP binds directly to Ad5E1A. (i) [35S]methionine- labelled CtIP or (ii) [35S]methionine-labelled CtBP1 were incubated with GST-Ad5E1A proteins and polypeptides (25 mg) as shown by Coomassie Blue staining (iii). Bound CtIP or CtBP1 were fractioned by SDS–polyacrylamide gel electrophoresis and detected by fluorography and autoradiography. Input represents 5% of the amount added in the pull downs. AdE1A, adenovirus onco- protein early region 1A; CtBP1, C-terminal-binding protein; CtIP, C-terminal-binding protein interacting protein; GST, glutathione- S-transferase.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Incubation, Staining, Polyacrylamide Gel Electrophoresis, Autoradiography, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 4 Mapping the CtIP-binding site in Ad5E1A. (a) Dimen- sions of GST-Ad5E1A polypeptides used in the pull-down assays. (b) [35S]methionine-labelled CtIP was incubated with GST-Ad5E1A polypeptides (25 mg) as shown. A Coomassie Blue stained gel of the GST fragments is shown in the lower panel. (c) [35S]methionine CtIP, p107 and TBP were incubated with GST-Ad512SE1A, GST- Ad513SE1A and GST-Ad5CR3. Bound proteins were fractioned by SDS–polyacrylamide gel electrophoresis and visualized by fluorography and autoradiography. Densitometric scanning was used to quantify the proportion of each of the proteins bound. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein; GST, glutathione- S-transferase.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Binding Assay, Incubation, Staining, Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 5 Analysis of the binding site for CtIP in the N-terminal region of Ad5E1A. (a) [35S]methionine-labelled CtIP was incubated with GST-Ad512SE1A proteins carrying the amino-acid substitutions shown (20 mg). Bound proteins were isolated with glutathione agarose, eluted, fractionated by SDS–PAGE and detected by fluorography and autoradiography. Binding to wt Ad512S and Ad513S E1A is also shown. Input represents 5% of the total added in the pull-downs. Coomassie Blue-stained gels of the GST-AdE1As are shown. (b) CtIP binding to E1A CR3 from different viral serotypes. [35S]methionine-labelled CtIP was incubated with GST fusion proteins containing the CR3 regions of AdE1As, from different viral serotypes (25 mg). Bound proteins were fractioned by SDS–PAGE and detected by fluorography and autoradiography. Input represents 10% of the total added. A Coomassie Blue-stained gel of the GST-CR3 proteins is shown in the lower panel. AdE1A, adenovirus oncoprotein early region 1A; CR, conserved region; CtIP, C-terminal-binding protein interacting protein; GST, glutathione-S-transferase; SDS–PAGE, sodium dodecylsulphate–polyacrylamide gel electrophoresis.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Binding Assay, Incubation, Isolation, SDS Page, Autoradiography, Staining, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 6 Complex formation of Ad5E1A, CtBP1 and CtIP. GST- Ad5E1A or GST-CtBP2 was incubated with [35S]-labelled CtIP in the presence of increasing concentrations of a peptide containing the PLDLS motif. Protein mixtures were incubated with glu- tathione agarose beads. The level of bound GST-Ad5E1A or GST- CtBP2 is shown by SDS–PAGE and staining for total protein (i). Bound [35S]-CtIP was detected by fluorography and autoradio- graphy (ii). AdE1A, adenovirus oncoprotein early region 1A; CtBP2, C-terminal-binding protein; CtIP, C-terminal-binding protein interacting protein; GST, glutathione-S-transferase.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Incubation, SDS Page, Staining, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 7 The effect of CtIP on Ad5E1A transactivation. CtIP or CtBP expression was reduced in A549 cells using appropriate siRNAs. Control cells were transfected with a nonspecific control siRNA. After 3 days, cells were transfected with pcDNA 3-Gal4 DBD and a Gal4-responsive luciferase reporter, pcDNA-Gal4 DBD-CR3 and a Gal4-responsive luciferase reporter or VP16 DBD and a Gal4-responsive luciferase reporter. (a) At 24 h post- transfection luciferase activity was determined. (b) Western blot showing reduction of CtIP expression (upper panel) and CtBP expression (lower panel) by siRNA. AdE1A, adenovirus oncopro- tein early region 1A; CtBP2, C-terminal-binding protein; CtIP, C- terminal-binding protein interacting protein; DBD, DNA-binding domain; GST, glutathione-S-transferase; siRNA, small interfering RNA.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Expressing, Control, Transfection, Luciferase, Activity Assay, Western Blot, Binding Assay, Small Interfering RNA

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 9 Ad5E1A disrupts complexes between CtIP and Rb and p130. (a) MCF7 cells were mock infected or infected with dl1520 (20 pfu/cell) for 24 h. CtIP was immunoprecipitated using a goat antibody. After fractionation by SDS–polyacrylamide gel electro- phoresis samples were western blotted for Rb or p130 as shown, (b) as for (a) except cells were infected with dl520 or dl1108 and blotted for Rb. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C- terminal-binding protein interacting protein; Rb, retinoblastoma.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Infection, Immunoprecipitation, Fractionation, Western Blot, Binding Assay

Journal: Oncogene

Article Title: C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3.

doi: 10.1038/sj.onc.1210551

Figure Lengend Snippet: Figure 10 Ad5E1A inhibits phosphorylation of CtIP. A549 cells and A549 cells stably expressing Ad512SE1A or Ad513SE1A were subjected to IR (20 Gy). Cells were harvested at the times shown and western blotted for CtIP and b-actin. Phosphorylated CtIP can be seen as a slower migrating band. AdE1A, adenovirus oncoprotein early region 1A; CtIP, C-terminal-binding protein interacting protein.

Article Snippet: Rb was detected with a mouse monoclonal antibody from Becton–Dickinson (Franklin Lakes, NJ, USA) and p107 (C18) and p130 (C20) with rabbit polyclonal antibodies (Santa Cruz) Ad5E1A was immunoprecipitated and detected in western blots using the mouse monoclonal antibody M58 (Becton-Dickinson).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Binding Assay

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: Aumolertinib inhibits proliferation, migration, and induces apoptosis in PC-9 cells. (A) CCK-8 assay was used to evaluate the inhibitory effects of aumolertinib on cell viability of PC-9 cells. (B) CCK-8 assay was used to evaluate the inhibitory effects of aumolertinib on proliferation of PC-9 cells (P=0.003). (C,D) In vitro wound healing assay was performed to assess the effects of aumolertinib on migration ability of PC-9 cells (P=0.02), magnification 100×. (E,F) Flow cytometry with Annexin V/PI staining was used to determine the effects of aumolertinib on apoptosis of PC-9 cells (P<0.001). (G,H) Flow cytometric analysis of the cell cycle in PC-9 cells treated with aumolertinib (P=0.049). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ETV4 , ETS variant transcription factor 4; FITC, fluorescein isothiocyanate; PI, propidium iodide; si ETV4 , ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Migration, CCK-8 Assay, In Vitro, Wound Healing Assay, Flow Cytometry, Staining, Cell Counting, Variant Assay, Negative Control, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: Silencing ETV4 potentiates aumolertinib’s growth inhibition in PC-9 cells. (A) PC-9 cells were transfected with si ETV4 (si ETV4 -1, si ETV4 -2, and si ETV4 -3) or siNC. Knockdown efficiency using si ETV4 in PC-9 cells was analyzed by qRT-PCR (P<0.001). (B,C) Knockdown efficiency using si ETV4 in PC-9 cells was analyzed by immunoblotting. GAPDH was used as the control (P<0.001). (D) CCK-8 assay was used to evaluate the effects of si ETV4 on proliferation of PC-9 cells that treated with aumolertinib (P=0.001). **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ETV4 , ETS variant transcription factor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; si ETV4 , ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Inhibition, Transfection, Knockdown, Quantitative RT-PCR, Western Blot, Control, CCK-8 Assay, Cell Counting, Variant Assay, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: ETV4 knockdown enhances aumolertinib’s anti-tumor effects in PC-9 cells. (A,B) Wound healing assay was performed to evaluate the effect of ETV4 knockdown on migration ability of PC-9 cells that treated with aumolertinib (P=0.03), magnification 100×. (C,D) Flow cytometry with Annexin V/PI staining was used to assess the influence of ETV4 knockdown on apoptosis of PC-9 cells that treated with aumolertinib (P<0.001). (E,F) Flow cytometric analysis was used to assess the influence of ETV4 knockdown on cell cycle of PC-9 cells that treated with aumolertinib. *, P<0.05; **, P<0.01; ***, P<0.001. ETV4 , ETS variant transcription factor 4; FITC, fluorescein isothiocyanate; PI, propidium iodide; si ETV4 , ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Knockdown, Wound Healing Assay, Migration, Flow Cytometry, Staining, Variant Assay, Negative Control, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: ETV4 enhances the inhibitory effects of aumolertinib on tumor growth in vivo . (A) Photographs of mice models treated with ETV4 inhibitors and/or aumolertinib. (B) Tumor growth curves of mice models treated with ETV4 inhibitors and/or aumolertinib (P<0.001). Tumor volume (mm 3 ) was measured every 3 days. (C,D) Comparison of tumor weights (g) treated with ETV4 inhibitors and/or aumolertinib (P<0.001). *, P<0.05; ***, P<0.001. ALM, aumolertinib administration; ETV4 , ETS variant transcription factor 4; NC, negative control; si ETV4 , ETV4 siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: In Vivo, Comparison, Variant Assay, Negative Control, Small Interfering RNA

Journal: Oncotarget

Article Title: ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor

doi: 10.18632/oncotarget.23173

Figure Lengend Snippet: (A) RNA was isolated from 55 freshly frozen human GISTs and analyzed for ETV4 and ETV1 using real-time PCR. Prim/UT–primary, untreated GIST, Met/Res – metastatic, imatinib-resistant GIST, HPF – high power field. Horizontal bars represent the median (left and right). Student’s t test, * P < 0.05. A scatter plot shows the correlation between ETV4 and mitotic count (middle) (Spearman’s rho = 0.67 with P = 0.001 per 2-tailed test). (B) Protein extracts were prepared from freshly frozen human GISTs that were either primary, untreated tumors with a mitotic rate of ≤5/50 HPF or >5/50 HPF, and metastatic, imatinib-resistant GISTs with a mitotic rate of >10/50 HPF and immunoblotted with anti-human ETV4 IgG followed by anti-GAPDH IgG. (C) Representative ETV4 staining from 46 paraffin-embedded human GISTs. Scale bar, 20 μm. Inset is 40x magnification to show nuclear staining. (D) Freshly isolated KIT - and KIT + cells from 3 metastatic, imatinib-resistant human GISTs with high ETV4 expression were analyzed for ETV4 mRNA by real-time PCR. Bars, mean ± SEM. Student’s t test, *** P <0.001.

Article Snippet: To generate cells transiently overexpressing ETV4, GIST T1 or GIST882 cells were transfected with either control plasmid (pCMV6-ctrl, PS10001, Origene) or human ETV4 plasmid (pCMV6-ETV4, RC202010, Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Staining, Expressing

Journal: Oncotarget

Article Title: ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor

doi: 10.18632/oncotarget.23173

Figure Lengend Snippet: (A) GFP expression in GIST882 cells after transduction with either ETV4 or control shRNA lentiviral constructs and growth under continuous puromycin selection. (B) ETV4 and ETV1 mRNA expression measured by real-time PCR. (C) Cell viability of GIST882 cells after ETV4 knockdown. (D) Matrigel invasion assay of GIST882 cells after ETV4 knockdown. Scale bar, 100 μm. (E) 1x10 5 GIST882 cells stably transduced with ETV4 or control shRNA were injected into the flanks of NSG mice and tumors were harvested 4 months later. Representative gross pictures and tumor weights are shown. n = 8 mice per group. Scale bar, 1 cm. (F) Ki-67 staining in ETV4 knockdown or control tumors. Scale bar, 20 μm. (G) Cell viability of murine S2 cells after stable ETV4 knockdown by shRNA infection. (H) Matrigel invasion assay of murine S2 cells after ETV4 knockdown. (I) Weights of flank tumors in NSG mice 4 months after inoculation with 1x10 5 S2 cells transduced with ETV4 or control shRNA (n = 4 mice per group). (J) Representative Ki-67 staining in ETV4 knockdown or control S2 tumors. Scale bar, 20 μm. Lines represent the median. All bars, mean ± SEM, Student’s t test; * P < 0.05, *** P < 0.001.

Article Snippet: To generate cells transiently overexpressing ETV4, GIST T1 or GIST882 cells were transfected with either control plasmid (pCMV6-ctrl, PS10001, Origene) or human ETV4 plasmid (pCMV6-ETV4, RC202010, Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Expressing, Transduction, Control, shRNA, Construct, Selection, Real-time Polymerase Chain Reaction, Knockdown, Invasion Assay, Stable Transfection, Injection, Staining, Infection

Journal: Oncotarget

Article Title: ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor

doi: 10.18632/oncotarget.23173

Figure Lengend Snippet: (A) Human GIST T1 cells transfected with ETV4 siRNA, control siRNA, or ETV1 SMARTpool siRNA were assayed for ETV4 or ETV1 expression by real-time PCR. (B) Transcriptome analysis of GIST T1 cells following transfection with either ETV4 siRNA or control siRNA for 48h (n=2/group). (C) Real-time PCR for CDKN1C and SFRP4 mRNA. (D) Analysis of cell cycle profile in GIST T1 cells after 48h transfection with control siRNA or ETV4 siRNA. (E) Western blot of GIST T1 cells following transfection with either ETV4 or control siRNA for 96h. Bar graph shows densitometry analysis of the relative expression normalized to GAPDH when compared to control (neg) siRNA. (F) Western blot of GIST882 cells following stable transduction with either ETV4 shRNA or control shRNA. All bars, mean ± SEM. Student’s t test; * P < 0.05.

Article Snippet: To generate cells transiently overexpressing ETV4, GIST T1 or GIST882 cells were transfected with either control plasmid (pCMV6-ctrl, PS10001, Origene) or human ETV4 plasmid (pCMV6-ETV4, RC202010, Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transduction, shRNA

Journal: Oncotarget

Article Title: ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor

doi: 10.18632/oncotarget.23173

Figure Lengend Snippet: (A) Western blot of nuclear and cytoplasmic extracts from GIST T1 cells that had been transfected with control or ETV4 expression plasmid for 48h. (B) Real-time PCR of CCND1 (cyclin D1) and Myc mRNA expression in GIST T1 cells after 48h transfection with control or ETV4 expression plasmid. (C) GIST T1 cells were transfected with the indicated constructs for 48h. The nuclear extracts were used for western blot. (D) Real time PCR of ETV4 mRNA expression in GIST882 cells after 48h transfection with control or ETV4 expression plasmid. (E) GIST882 cells were transfected with either control or human ETV4 plasmids for 48h, and then cells were treated with or without 125 ng/ml rhWnt3a overnight. Total protein extracts were analyzed by western blot as indicated. All bars, mean ± SEM. Student’s t test; * P < 0.05.

Article Snippet: To generate cells transiently overexpressing ETV4, GIST T1 or GIST882 cells were transfected with either control plasmid (pCMV6-ctrl, PS10001, Origene) or human ETV4 plasmid (pCMV6-ETV4, RC202010, Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Western Blot, Transfection, Control, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Construct

Journal: Oncotarget

Article Title: ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor

doi: 10.18632/oncotarget.23173

Figure Lengend Snippet: (A) GIST T1 cells were transfected with either ETV4 siRNA or control siRNA for 72h, and then cells were treated with MG132 10 μM for 6h prior to harvest. Nuclear protein extracts were analyzed by western blot as indicated. (B) GIST T1 cells were transfected with control or human ETV4 plasmids for 48h and treated with MG132 10 μM overnight before harvesting. Nuclear protein extracts were analyzed by western blot as indicated. (C) Representative CHX-chase assays (of 2 performed) to determine the stability (half-life) of nuclear β-catenin in GIST T1 cells 48h after transfection with control siRNA or ETV4 siRNA. Cells were collected after the addition of 200 mg/ml CHX at the indicated time points. Relative nuclear β-catenin levels were determined by normalizing to the loading control (lamin B1) and then normalizing to the t = 0h control siRNA. Immunoblots of nuclear extracts are shown. (D) GIST T1 cells were transfected with the indicated siRNA and harvested 48h later and nuclear extracts were analyzed by western blot. (E) GIST T1 cells were transfected with control or ETV4 siRNA for 72h and then treated with MG132 10 μM for 6h prior to harvest. Nuclear extracts were immunoprecipitated by either anti-β-catenin or anti-COP1 and western blot was performed as indicated. (F) GIST T1 cells were transfected with the indicated constructs and harvested 48h later and nuclear extracts were analyzed by western blot.

Article Snippet: To generate cells transiently overexpressing ETV4, GIST T1 or GIST882 cells were transfected with either control plasmid (pCMV6-ctrl, PS10001, Origene) or human ETV4 plasmid (pCMV6-ETV4, RC202010, Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, Construct

Journal: Oncotarget

Article Title: ETV4 collaborates with Wnt/β-catenin signaling to alter cell cycle activity and promote tumor aggressiveness in gastrointestinal stromal tumor

doi: 10.18632/oncotarget.23173

Figure Lengend Snippet: GSEA was performed on high throughput RNA sequencing data from 36 selected human GISTs. (A) Enrichment of cell cycle genes and (B) negative regulation of Wnt genes was found in Met/Res (>10/50 HPF) GISTs (N = 22) compared to Prim/UT (<5/50 HPF) GISTs (N = 14). False Discovery Rate (FDR) and Normalized Enrichment Score (NES) were indicated. (C) The relative mRNA expression of CDC20 and DKK4 was analyzed by real-time PCR in ETV4-low and high GIST tumors. Horizontal bars represent the median. Prim/UT (<5/50 HPF) – primary, untreated GIST (n = 13), Met/Res (>5/50 HPF) – metastatic, imatinib-resistant GIST (n = 15). Student’s t test; * P < 0.05. (D) Western blot of Wnt pathway components in ETV4-high (metastatic, imatinib-resistant GISTs (Met/Res) with a mitotic rate >10/50 HPF) or ETV4-low (primary, untreated GISTs (Prim/UT) with a mitotic rate ≤5/50 HPF). (E) Representative staining of β-catenin and ETV4 expression in 46 GIST specimens. Staining intensity was scored as negative to weak (0), moderate (1+), or high (2+). Scale bar, 20 μm. (F) Correlation of ETV4 and β-catenin expression staining in 46 GISTs. Spearman’s rho = 0.77 with P = 0.001 per 2-tailed t test. (G) Real-time PCR showing relative mRNA expression of ETV4 in four PDXs compared to GIST T1 xenografts. All bars, means ± SEM. Student’s t test; * P < 0.05. (H) Immunoblots of nuclear extracts from GIST T1 xenografts and four PDXs. PDX-1: KIT exon 11/13 mutation; PDX-2: WT KIT with SDHB/KRAS mutation; PDX-3: KIT exon 9/11 mutation; PDX-4: KIT exon 11 mutation with CDKN2A deletion. All four PDXs are from tumors of metastatic, imatinib-resistant GIST patients with a high mitotic rate.

Article Snippet: To generate cells transiently overexpressing ETV4, GIST T1 or GIST882 cells were transfected with either control plasmid (pCMV6-ctrl, PS10001, Origene) or human ETV4 plasmid (pCMV6-ETV4, RC202010, Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: High Throughput Screening Assay, RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Mutagenesis

Journal: bioRxiv

Article Title: A single-component optogenetic toolkit for programmable control of microtubule

doi: 10.1101/2025.10.31.685931

Figure Lengend Snippet: (a) Domain organization of mouse KIF5A (mKIF5A), showing the N-terminal motor head, central coiled-coil stalk, and C-terminal cargo-binding tail. (b) Schematic of mKIF5A truncation variants fused to CRY2 for optogenetic activation. Variant V4 (residues 1-379) was identified as the optimal construct and designated OptoMotor. (c) Design principle of OptoMotor. Blue light illumination induces CRY2 oligomerization to drive reassembly of the truncated motor complex and restoring plus-end-directed motility along under MTs. (d) Confocal images of selected CRY2-KIF5A truncation variants expressed in HeLa cells under dark and illuminated conditions. Variant V1 exhibited constitutive peripheral accumulation; V4 showed robust light-induced redistribution to the cell periphery without appreciable basal activity; and V6 displayed pronounced MT labeling. See Supplementary Fig. 7 for the complete set of variants. (e) Quantification of periphery-to-cytosol fluorescence ratios across truncation variants. Each symbol represents the mean of 5-6 cells from a single imaging field. A total of 40-50 cells were analyzed per construct across three independent biological replicates. (f) Time-lapse images showing reversible distribution of OptoMotor during repeated dark-light cycles. Also see Supplementary Video 11 . (g) Kinetic trace of peripheral intensity changes showing OptoMotor activation (t 1/2 , ON = 0.9 min) and deactivation (t 1/2 , OFF = 3.7 min). (h) Localized blue light illumination within defined regions of interest (ROIs R1, R2) elicited spatially confined redistribution of OptoMotor in single cells. (i) Quantification of relative fluorescence intensity changes within ROIs confirming high spatial precision of light-triggered activation. (j) Schematic of the chimeric OptoMotor-Tail construct, generated by fusing the cargo-binding C-terminal tail of mKIF5A (residues 907-1027) to OptoMotor. Upon blue light activation, OptoMotor-Tail drives peripheral transport of lysosomes to enhance mTORC signaling. (k) Confocal images of HeLa cells co-expressing LAMP1-GFP and mCh-OptoMotor-Tail showing light-induced redistribution of lysosomes toward the cell periphery. (l) Immunoblot showing phosphorylation of S6K at T389 (P-S6K) as a readout of mTORC1 activity. Blue light illumination enhanced P-S6K levels in cells expressing OptoMotor-Tail, consistent with lysosomal repositioning mediated activation of mTORC1 signaling. Starved cells before and after nutrient recovery were used as negative and positive controls for reporting mTORC1 activity.

Article Snippet: The plasmid templates for EB1 (#17234), CLIP170 (#54044), CAMSAP1 (# 59036),CAMSAP2 (#59037), KIF5A (#166954), and spastin (#134461) were purchased from Addgene.

Techniques: Binding Assay, Activation Assay, Variant Assay, Construct, Activity Assay, Labeling, Fluorescence, Imaging, Generated, Expressing, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

doi: 10.1038/srep28241

Figure Lengend Snippet: ( a ) Co-IP of myc-E1A with DYRK1A, DYRK1B, HIPK2 and DCAF7. - HeLa cells were transfected to co-express myc-E1A (289 amino acid form) GFP-DYRK1A, DYRK1B or HIPK2 and either FLAG-DCAF7 or a control vector. The vertical line indicates where irrelevant lanes were deleted from the final image. Note that GFP-HIPK2 is difficult to reveal on the blots due to its large size and could not be detected in the cell lysates (input). ( b ) Co-IP of endogenous DYRK1A/DCAF7 with E1A. - The lysate of untransfected HEK293 cells or HeLa cells was subjected to IP with either goat anti DYRK1A (αD1A) or mouse anti-E1A (αE1A). An unrelated goat antibody (Ctrl) was used as a negative control for the IP with αD1A. HeLa cells (which lack E1A) were used as background control for the αE1A IP. The heavy chain of the immunoprecipitating E1A antibody is marked by an asterisk (IgG). ( c ) DYRK1A, DCAF7 and E1A are components of a common complex. - HEK293-(GFP-DYRK1A-tetOn) cells were transfected with a FLAG-DCAF7 expression vector or empty control plasmid and induced with doxycyclin to express GFP-DYRK1A. Lysates were subjected to sequential IP with anti FLAG and anti GFP. The two E1A bands may correspond to the major protein forms of 289 and 243 amino acids that are expressed in HEK293 cells. ( d ) GST pulldown assay. - HeLa cells were transfected to express GFP or GFP-DYRK1A either with FLAG-DCAF7 or alone. Aliquots of cell lysates were used for pulldown assays with agarose-bound GST or a GST-tagged construct of the exon2-encoded portion of E1A (E1A-X2) as bait. A deletion mutant (X2Δ, deletion of amino acids 255–270) that lacks the DYRK1A/DCAF7 binding region of E1A served as negative control. ( e ) Outline of the pulldown assay ( f , g ) In vitro interaction assays. – GFP-DCAF7 was in vitro -translated in rabbit reticulocyte lysate (RRL) and used as a prey for GST-pulldown assays with immobilized GST fusion proteins as indicated below the bottom panel. In parallel control samples (Co), in vitro transcription was driven by the empty vector.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-DCAF7 (EPR8712, Epitomics/Abcam), rabbit polyclonal anti GFP (Clontech 632592), goat polyclonal anti-GFP (Rockland) and anti-c-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-DYRK1A (clone 7D10, Abnova, directed against a C-terminal epitope), anti FLAG (clone M2, Sigma), anti HA (clone 3F10, Roche) and anti E1A-pSer219 (C-9, sc-374663, Santa Cruz Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Transfection, Control, Plasmid Preparation, Negative Control, Expressing, GST Pulldown Assay, Construct, Mutagenesis, Binding Assay, In Vitro

Journal: Scientific Reports

Article Title: The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

doi: 10.1038/srep28241

Figure Lengend Snippet: Human HT1080 cells were treated with control siRNA, siRNA specific to DYRK1A ( a ) or siRNA specific to DCAF7 ( b ). Cells were subsequently co-transfected with the blank vector, GFP-E1A (WT, 289 amino acid form) or the E1A point mutant R262/263E (Mu) and vectors expressing FLAG-DCAF7 ( a ) or HA-DYRK1A ( b ). The E1A-R262/263E mutant does not interact with DYRK1A or DCAF7 and served as a specificity control. Lysates were immunoprecipitated using anti-FLAG antibodies ( a ) or anti-HA antibodies ( b ) and immunoblotted using anti-GFP antibodies to detect the presence of co-precipitating E1A.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-DCAF7 (EPR8712, Epitomics/Abcam), rabbit polyclonal anti GFP (Clontech 632592), goat polyclonal anti-GFP (Rockland) and anti-c-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-DYRK1A (clone 7D10, Abnova, directed against a C-terminal epitope), anti FLAG (clone M2, Sigma), anti HA (clone 3F10, Roche) and anti E1A-pSer219 (C-9, sc-374663, Santa Cruz Biotechnology).

Techniques: Control, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Immunoprecipitation

Journal: Scientific Reports

Article Title: The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

doi: 10.1038/srep28241

Figure Lengend Snippet: ( a , b ) Co-IP of myc-E1A wild type DYRK1A, deletion mutants of DYRK1A and a kinase-negative point mutant of DYRK1A (K188R). - Lysates of HeLa cells expressing myc-E1A, FLAG-DCAF7 and the indicated GFP-DYRK1A constructs were subjected to anti GFP IP. The dashed lines indicate where irrelevant regions of the blots were deleted from the final image. ( c , d ) GST pulldown assays. - HeLa cells were transfected to co-express FLAG-DCAF7 with GFP-DYRK1A constructs or GFP-HIPK2 constructs as indicated. Cell lysates were subjected to GST-pulldown assay with immobilized GST or GST-E1A-X2 and bound proteins were analysed by immunoblotting. GST-E1A-X2Δ lacks the DYRK1A/DCAF7 binding region of E1A and served as negative control. To reveal the GFP-DYRK1A 1–176 construct, a polyclonal goat antibody directed against an N-terminal epitope was used in panel ( c ). This antibody crossreacts with an unidentified band (marked by an asterisk) that is not detected by the monoclonal DYRK1A antibody used in . Endogenous DYRK1A is detected as a double band at ~90 kDa.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-DCAF7 (EPR8712, Epitomics/Abcam), rabbit polyclonal anti GFP (Clontech 632592), goat polyclonal anti-GFP (Rockland) and anti-c-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-DYRK1A (clone 7D10, Abnova, directed against a C-terminal epitope), anti FLAG (clone M2, Sigma), anti HA (clone 3F10, Roche) and anti E1A-pSer219 (C-9, sc-374663, Santa Cruz Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, Expressing, Construct, Transfection, GST Pulldown Assay, Western Blot, Binding Assay, Negative Control

Journal: Scientific Reports

Article Title: The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

doi: 10.1038/srep28241

Figure Lengend Snippet: ( a ) In vitro kinase assay. - Bacterially expressed GST-E1A-X2 was incubated with recombinant DYRK1A at 30 °C in the presence of 1 mM ATP. Aliquot of the reaction were taken at variable times and phosphorylation of E1A was detected by Western blot analysis with a phosphospecific antibody directed against pSer219. ( b ) Phosphorylation of E1A in HEK293 cells. - HEK293-(GFP-DYRK1A-tetOn) cells were transfected with a FLAG-DCAF7 expression vector or empty control plasmid and either induced with doxycyclin (dox) to express GFP-DYRK1A or not induced. Two days after transfection, total cellular lysates were analyzed for Ser219 phosphorylation. Detection of E1A by the monoclonal antibody M58 is independent of the phosphorylation state. The asterisks mark an upshifted band in DYRK1A overexpressing samples. ( c – e ) Phosphorylation of E1A exon2 by DYRK1A and HIPK2. - HeLa cells were co-transfected with expression plasmids for myc-E1A-X2, FLAG-DCAF7 and DYRK1A, HIPK1, HIPK2 or mutant kinase constructs as indicated. In c , myc-E1A-X2 was immunoprecipitated and either dephosphorylated by calf intestinal phosphatase (CIP) or not treated before SDS-PAGE. The asterisks mark the light chain bands of the immunoprecipitating antibody. In ( d , e ), total cellular lysates were analysed for phosphorylation of E1A-X2 2 days after transfection.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-DCAF7 (EPR8712, Epitomics/Abcam), rabbit polyclonal anti GFP (Clontech 632592), goat polyclonal anti-GFP (Rockland) and anti-c-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-DYRK1A (clone 7D10, Abnova, directed against a C-terminal epitope), anti FLAG (clone M2, Sigma), anti HA (clone 3F10, Roche) and anti E1A-pSer219 (C-9, sc-374663, Santa Cruz Biotechnology).

Techniques: In Vitro, Kinase Assay, Incubation, Recombinant, Phospho-proteomics, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, Mutagenesis, Construct, Immunoprecipitation, SDS Page

Journal: Scientific Reports

Article Title: The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

doi: 10.1038/srep28241

Figure Lengend Snippet: HeLa cells transiently transfected to co-express either wild type GFP-DYRK1A (WT) or GFP-DYRK1A-Δ93–104 together with E1A (243 amino acid form) and/or FLAG-DCAF7. Proteins were detected by autofluorescence (GFP) or immunofluorescence (E1A, FLAG-DCAF7). Nuclei were stained with DAPI. Scale bar, 100 μm.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-DCAF7 (EPR8712, Epitomics/Abcam), rabbit polyclonal anti GFP (Clontech 632592), goat polyclonal anti-GFP (Rockland) and anti-c-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-DYRK1A (clone 7D10, Abnova, directed against a C-terminal epitope), anti FLAG (clone M2, Sigma), anti HA (clone 3F10, Roche) and anti E1A-pSer219 (C-9, sc-374663, Santa Cruz Biotechnology).

Techniques: Transfection, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

doi: 10.1038/srep28241

Figure Lengend Snippet: Multiple binding sites allow for the simultaneous interaction of DCAF7 with E1A and DYRK1. E1A is an intrinsically disordered protein that is known to interact with many cellular proteins including the pocket proteins (RB1, RBL1). NLS, nuclear localization signal.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-DCAF7 (EPR8712, Epitomics/Abcam), rabbit polyclonal anti GFP (Clontech 632592), goat polyclonal anti-GFP (Rockland) and anti-c-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-DYRK1A (clone 7D10, Abnova, directed against a C-terminal epitope), anti FLAG (clone M2, Sigma), anti HA (clone 3F10, Roche) and anti E1A-pSer219 (C-9, sc-374663, Santa Cruz Biotechnology).

Techniques: Binding Assay