Journal: The Journal of Biological Chemistry

Article Title: Characterizing the protein–protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization

doi: 10.1016/j.jbc.2024.105651

Figure Lengend Snippet: Biophysical investigation of the 14-3-3σ/MDM2 PPI. A and B , FP data for MDM2 peptide binding to 14-3-3σ ( A ) and 14-3-3ζ ( B , the second binding event is shown in gray ). 14-3-3 protein was titrated to 10 nM FITC-labeled peptide in buffer containing 25 mM Hepes pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 0.1% v/v Tween20, 0.1 mg/ml BSA, and 1% v/v DMSO. Error bars represent SD for n = 3 replicates. C and D , ITC data for MDM2 peptide binding to 14-3-3σ ( C ) and 14-3-3ζ ( D ). Acetylated MDM2 161-191 pS166/pS186 peptide (1.0 mM) was titrated to 14-3-3 protein (0.1 mM) at 25 °C in buffer containing 25 mM Hepes pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , and 1% v/v DMSO. The TFA content of the peptide was determined by 19 F-NMR in order to accurately calculate peptide concentration (see Fig. S5 and for details). E , native mass spectrometry data confirming 2:1 stoichiometry of 14-3-3σ monomer to MDM2 peptide. All protein concentrations are given as 14-3-3 monomer concentrations. FP, fluorescence polarization; ITC, isothermal titration calorimetry; MDM2, Mouse Double Minute 2; PPI, protein–protein interaction.

Article Snippet: ITC experiments were conducted using the Malvern MicroCal ITC200 instrument (monophosphorylated peptides) or Malvern PEAQ ITC instrument (di-phosphorylated peptide).

Techniques: Binding Assay, Labeling, Concentration Assay, Mass Spectrometry, Fluorescence, Isothermal Titration Calorimetry

Journal: The Journal of Biological Chemistry

Article Title: Characterizing the protein–protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization

doi: 10.1016/j.jbc.2024.105651

Figure Lengend Snippet: K d values and thermodynamic parameters for MDM2 161-191 pS166/pS186 peptide binding to 14-3-3σ and 14-3-3ζ

Article Snippet: ITC experiments were conducted using the Malvern MicroCal ITC200 instrument (monophosphorylated peptides) or Malvern PEAQ ITC instrument (di-phosphorylated peptide).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterizing the protein–protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization

doi: 10.1016/j.jbc.2024.105651

Figure Lengend Snippet: Stabilization of the 14-3-3/MDM2 PPI by FC-A. A , FP data for MDM2 161-191 pS166/pS186 peptide binding to 14-3-3σ in the presence of FC-A. 14-3-3σ was titrated to 10 nM FITC-labeled peptide and increasing fixed concentrations of FC-A. B , FP dose-response data for FC-A. FC-A was titrated to fixed concentration of 14-3-3σ (1 μM) and FITC-labeled peptide (10 nM). C , FP data for MDM2 159-173 pS166 and MDM2 179-193 pS186 peptide binding to 14-3-3σ. 14-3-3σ was titrated to 10 nM FITC-labeled peptide and 1.0 mM FC-A. All FP experiments were conducted in buffer containing 25 mM Hepes pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 0.1% v/v Tween20, 0.1 mg/ml BSA, and 1% v/v DMSO. Error bars represent SD for n = 3 replicates. D , ITC data for MDM2 161-191 pS166/pS186 peptide binding to 14-3-3σ in the presence of FC-A. Acetylated MDM2 161-191 pS166/pS186 peptide (1.0 mM) was titrated to 14-3-3σ protein (0.1 mM) in the presence of FC-A (1.0 mM) at 25 °C in buffer containing 25 mM Hepes pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , and 1% v/v DMSO. E , native mass spectrometry data showing the FC-A concentration-dependent formation of a ternary complex. Red arrows highlight peaks for the ternary complex. Error bars represent SD for n = 3 replicates. FP, fluorescence polarization; ITC, isothermal titration calorimetry; MDM2, Mouse Double Minute 2; PPI, protein–protein interaction.

Article Snippet: ITC experiments were conducted using the Malvern MicroCal ITC200 instrument (monophosphorylated peptides) or Malvern PEAQ ITC instrument (di-phosphorylated peptide).

Techniques: Binding Assay, Labeling, Concentration Assay, Mass Spectrometry, Fluorescence, Isothermal Titration Calorimetry

Journal: Cancer & metabolism

Article Title: ETV4/ALYREF-mediated glycolytic metabolism through PKM2 enhances resistance to ferroptosis and promotes the development of intrahepatic cholangiocarcinoma.

doi: 10.1186/s40170-025-00387-1

Figure Lengend Snippet: Fig. 1 ETV4 and PKM2 enhance ICC cell proliferation and glycolytic metabolism. (A) High expression levels of ETV4 and PKM2 in ICC were analyzed using the TCGA database. (B) The analysis of the survival rate in the presence of ETV4 and PKM2. (C) The expression levels of ETV4 and PKM2 were detected by WB. (D) Cell proliferation was measured by CCK-8 assay. (E) ICC cell proliferation was detected by the EdU assay (Magnification: ×50, scale bar = 200 μm). (F) Cell migration of ICC cells was assessed by Transwell assay (Magnification: ×100, scale bar = 100 μm). (G-I) Measurement of the GLU, LD, and ATP con tent in ICC cells. (J) Expression of PDK1, LDHA, and GLUT1 was assessed by WB. (K) Bioinformatics prediction of binding sites between ETV4 and PKM2. (L) ChIP validation of the binding of ETV4 and the promoter of ALYREF. *P < 0.05; **P < 0.01; ***P < 0.001; n = 3

Article Snippet: Following the instructions provided by the ChIP assay kit (ab500; Abcam), sequential steps were performed, including cross-linking, sonication, detection of DNA fragment Table 1 The information on antibody Name Dilution rate Cat number Source Company Country ETV4 1:1000 AWA45623 Rabbit Abiowel China PKM2 1:2000 AWA43705 Rabbit Abiowell China ALYREF 1:1000 AWA51025 Rabbit Abiowell China PDK1 1:1000 ab202468 Rabbit Abcam USA LDHA 1:1000 19987-1-AP Rabbit Proteintech USA GLUT1 1:1000 ab115730 Rabbit Abcam USA NSUN2 1:1000 ab259941 Rabbit Abcam USA β-actin 1:5000 60008-1-Ig Mouse Proteintech USA HRP goat anti- mouse IgG (H + L) 1:5000 SA00001-1 / Proteintech USA HRP goat anti- Rabbit IgG (H + L) 1:5000 SA00001-2 / Proteintech USA lengths, immunoprecipitation, de-crosslinking, and DNA purification.

Techniques: Expressing, CCK-8 Assay, EdU Assay, Migration, Transwell Assay, Binding Assay, Biomarker Discovery

Journal: Cancer & metabolism

Article Title: ETV4/ALYREF-mediated glycolytic metabolism through PKM2 enhances resistance to ferroptosis and promotes the development of intrahepatic cholangiocarcinoma.

doi: 10.1186/s40170-025-00387-1

Figure Lengend Snippet: Fig. 2 ETV4 promotes ICC cell resistance to ferroptosis through PKM2-mediated glycolytic metabolism. (A) The expression levels of ETV4 and PKM2 were detected by WB. (B) ICC cell proliferation was assessed by CCK-8 assay. (C) ICC cell proliferation was detected by the EdU assay (Magnification: ×50, scale bar = 200 μm). (D) ICC cell migration was examined by Transwell assay (Magnification: ×100, scale bar = 100 μm). (E-G) Measurement of GLU, LD, and ATP content in ICC cells. (H) The expression of PDK1, LDHA, and GLUT1 assessed by WB. (I) CCK-8 assay was used to screen Erastin concentration. (J) ICC cell proliferation was assessed by CCK-8 assay. (K) Transwell assay was used to evaluate ICC cell migration after erastin induction (Magnification: ×100, scale bar = 100 μm). (L) FCM was used to measure ICC cell lipid ROS levels. (M) ELISA was used to detect MDA and Fe2+ levels in ICC cells. *P < 0.05; **P < 0.01; ***P < 0.001; n = 3

Article Snippet: Following the instructions provided by the ChIP assay kit (ab500; Abcam), sequential steps were performed, including cross-linking, sonication, detection of DNA fragment Table 1 The information on antibody Name Dilution rate Cat number Source Company Country ETV4 1:1000 AWA45623 Rabbit Abiowel China PKM2 1:2000 AWA43705 Rabbit Abiowell China ALYREF 1:1000 AWA51025 Rabbit Abiowell China PDK1 1:1000 ab202468 Rabbit Abcam USA LDHA 1:1000 19987-1-AP Rabbit Proteintech USA GLUT1 1:1000 ab115730 Rabbit Abcam USA NSUN2 1:1000 ab259941 Rabbit Abcam USA β-actin 1:5000 60008-1-Ig Mouse Proteintech USA HRP goat anti- mouse IgG (H + L) 1:5000 SA00001-1 / Proteintech USA HRP goat anti- Rabbit IgG (H + L) 1:5000 SA00001-2 / Proteintech USA lengths, immunoprecipitation, de-crosslinking, and DNA purification.

Techniques: Expressing, CCK-8 Assay, EdU Assay, Migration, Transwell Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer & metabolism

Article Title: ETV4/ALYREF-mediated glycolytic metabolism through PKM2 enhances resistance to ferroptosis and promotes the development of intrahepatic cholangiocarcinoma.

doi: 10.1186/s40170-025-00387-1

Figure Lengend Snippet: Fig. 6 The ETV4/ALYREF axis promotes tumor proliferation and glycolytic metabolism in mice. (A) Images of subcutaneous xenograft in mice. (B) The effect of ETV4/ALYREF axis on subcutaneous xenograft volume. (C) The effect of ETV4/ALYREF axis on subcutaneous xenografts weight. (D) The effect of ETV4/ALYREF axis on the concentration of LD in subcutaneous xenografts. (E) WB analysis of the expression of PKM2 and its downstream targets in subcutaneous xenografts. *P < 0.05; **P < 0.01; ***P < 0.001; n = 6

Article Snippet: Following the instructions provided by the ChIP assay kit (ab500; Abcam), sequential steps were performed, including cross-linking, sonication, detection of DNA fragment Table 1 The information on antibody Name Dilution rate Cat number Source Company Country ETV4 1:1000 AWA45623 Rabbit Abiowel China PKM2 1:2000 AWA43705 Rabbit Abiowell China ALYREF 1:1000 AWA51025 Rabbit Abiowell China PDK1 1:1000 ab202468 Rabbit Abcam USA LDHA 1:1000 19987-1-AP Rabbit Proteintech USA GLUT1 1:1000 ab115730 Rabbit Abcam USA NSUN2 1:1000 ab259941 Rabbit Abcam USA β-actin 1:5000 60008-1-Ig Mouse Proteintech USA HRP goat anti- mouse IgG (H + L) 1:5000 SA00001-1 / Proteintech USA HRP goat anti- Rabbit IgG (H + L) 1:5000 SA00001-2 / Proteintech USA lengths, immunoprecipitation, de-crosslinking, and DNA purification.

Techniques: Concentration Assay, Expressing

Journal: Nutrients

Article Title: Birch Pollen Related Pear Allergy: A Single-Blind Oral Challenge TRIAL with 2 Pear Cultivars

doi: 10.3390/nu13041355

Figure Lengend Snippet: Western blots (WB) of ‘Cepuna’ and ‘Conference’ pear total protein concentrate using the patient serum. The WB control blot was exposed to buffer instead of serum. Patient numbering is indicated by #.

Article Snippet: In brief, 20 μg of pear protein concentrate was separated by SDS PAGE on Bolt TM 4–12% Bis-Tris Plus gels next to a Precision Plus Protein Dual Xtra Standard molecular weight marker (Bio-rad, Hercules, CA, USA) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) and either stained by Simply Blue safe stain (Thermo Fisher Scientific Inc.) or transferred to a 0.2 μm nitrocellulose membrane (LKB, Bromma, Sweden) by Tris-glycine buffer (25 mM Tris, 190 mM glycine, 0.1% SDS, 20% methanol) for 36 min at 70 V using a Criterion blotter (Bio-rad).

Techniques: Western Blot, Control