Journal: The FEBS journal

Article Title: Structural and biochemical analysis of Bacillus anthracis prephenate dehydrogenase reveals an unusual mode of inhibition by tyrosine via the ACT domain

doi: 10.1111/febs.15150

Figure Lengend Snippet: Melting temperatures (T m ) of BaPDH and its complexes as determined by protein thermal shift assay. All ligands were used at 10 mM final concentration. ns – non-significant.

Article Snippet: Protein thermal shift assay The thermal stability of BaPDH and its complexes was measured by the protein thermal shift assay performed with a C1000 thermal cycler supplemented with a CFX96 real-time system in a 96-well PCR plate (Bio-Rad, Hercules, CA, USA).

Techniques: Thermal Shift Assay, Concentration Assay, Control, Purification

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 6 PYK2 is a potential target of Entrectinib. (A) The binding model between Entrectinib (pink) and the crucial residues of PYK2 (purple). (B) The bind ing affinity between PYK2 and Entrectinib was analyzed by Microscale thermophoresis (MST). (C) Cellular Thermal Shift Assay and Western blot were performed to evaluate the stability of PYK2 after incubation with or without Entrectinib at different temperatures. (D) Drug Affinity Responsive Target Sta bility and Western blot were performed to evaluate the resistance of PYK2 to enzymatic hydrolysis. (E) The phosphorylation levels of PYK2 in cells treated by TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM) were analyzed by western blot. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. ###P < 0.001 versus control group. ****P < 0.0001 versus TGFβ2 group

Article Snippet: In brief, after dyeing with N-hydroxysuccinimide, the recombinant human PYK2 protein (Solarbio, China) was diluted and mixed with Entrectinib of different concentrations.

Techniques: Binding Assay, Microscale Thermophoresis, Thermal Shift Assay, Western Blot, Incubation, Phospho-proteomics, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 7 The knockdown of PYK2 inhibits the TGFβ2-induced EMT through a non-Smad signaling pathway. (A) The knockdown efficiency of PYK2 was evaluated by western blotting. (B) The knockdown of PYK2 inhibited the EMT induced by TGFβ2. The efficacy of Entrectinib is influenced by PYK2 knock down. (C) PYK2 knockdown inhibited the activation of AKT, JNK, ERK, and P38. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. #P < 0.05 and ##P < 0.01 versus the control group

Article Snippet: In brief, after dyeing with N-hydroxysuccinimide, the recombinant human PYK2 protein (Solarbio, China) was diluted and mixed with Entrectinib of different concentrations.

Techniques: Knockdown, Western Blot, Activation Assay, Control

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Elevated NAT10 expression correlates with poor prognosis in HCC patients. (A) Dot blot analyses of total RNA (5 µg) isolated from HCC tissues and adjacent noncancerous liver tissues using an anti‐ac4C antibody, with MB staining as loading control (left panel). Calculation of relative ac4C contents on RNA in HCC tissues and adjacent noncancerous liver tissues (right panel, N = 20). (B) Detection of ac4C levels on mRNA in the same 20 HCC tissues and adjacent noncancerous liver tissues by UPLC‐MS/MS analysis. (C) Large‐scale data mining was used to compare the expression differences in NAT10 mRNA between HCC tissues and adjacent noncancerous liver tissues. (D) Analysis of NAT10 protein levels in HCC and adjacent noncancerous liver tissues ( N = 165) using CPTAC data. (E) NAT10 protein levels in HCC tissues and adjacent noncancerous liver tissues measured by western blotting ( N = 8). (F) Representative IHC images of NAT10 expression in the Tongji cohort tissue microarray (TMA) including HCC tissues and adjacent noncancerous liver tissues (left panel); pie chart showing percentages of cases with differential NAT10 expression profiles compared to adjacent noncancerous liver tissues ( N = 103, right panel, scale bar: 200 µm or 20 µm). (G) Kaplan‐Meier plot correlating NAT10 expression with patient overall and recurrence‐free survival using CPTAC, TCGA, and Tongji datasets. (H) Analysis of NAT10 expression correlation with malignant features of HCC using Tongji TMA cohort and TCGA LIHC datasets. Statistical tests: (A, B, D) paired t test; (G) log‐rank test; (H) Pearson chi‐squared test (2‐sided). Abbreviations: ANL, adjacent noncancerous liver tissue; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; LIHC, Liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; CPTAC, Clinical Proteomic Tumor Analysis Consortium; MB, methylene blue; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; mRNA, messenger RNA.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Expressing, Dot Blot, Isolation, Staining, Control, Tandem Mass Spectroscopy, Western Blot, Microarray, Immunohistochemistry

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: NAT10 knockdown inhibits HCC progression in vitro and in vivo. (A) Western blot analysis of NAT10 expression post NAT10 knockdown (upper panel). Dot blot analysis of ac4C levels in total RNA and mRNA isolated from control or NAT10‐knockdown HCC cells (lower panel, MHCC‐97H and SNU449), with MB staining as loading control. (B) and (C) CCK8 assay for cell viability of NAT10‐knockdown and control cells at indicated time points. (D) Colony formation assay quantification for indicated cells. (E) Soft agar colony formation assay quantification for indicated cells. (F) Scratch wound healing assays quantification for indicated cells. (G) Cell migration and invasion assays quantification for MHCC‐97H and SNU449 cells. (H) Subcutaneous xenograft models with NAT10‐knockdown and control cells ( n = 5, scale bar: 1 cm). Tumor volume monitored and growth curves generated over 24 days (left panel); tumors weighed (right panel). (I) and (J) Representative images and quantitative data analysis of Ki67‐positive (I) and TUNEL‐positive (J) cells, (scale bar: 20 µm). (K) Representative bioluminescence imaging of liver orthotopic implantation models (left panel) and quantification of intrahepatic metastatic nodules (right panel) ( n = 5). (L) Representative bioluminescence imaging of lung metastasis models (left panel) and quantification of lung metastatic nodules (right panel) ( n = 5). (B‐G) Data shown as mean ± SD, n = 3. Statistical analysis: Unpaired t tests. ** P < 0.01, *** P < 0.001. Abbreviations: MB, methylene blue; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling; CCK‐8, Cell Counting Kit‐8; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Expressing, Dot Blot, Isolation, Control, Staining, CCK-8 Assay, Colony Assay, Soft Agar Assay, Migration, Generated, TUNEL Assay, Imaging, End Labeling, Cell Counting, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Effects of nat10 on ac4c mRNA modification and global mRNA translation. (A) Flow chart depicting acRIP‐seq. (B) Number of ac4C peaks identified in acRIP‐seq in shCtrl and shNAT10 MHCC‐97H cells. (C) Number of ac4C‐modified genes identified in acRIP‐seq. Common ac4C genes have ≥ 1 common ac4C peak, while unique ac4C genes have no common ac4C peaks. (D) Top consensus motif identified by HOMER with acRIP‐seq peaks in MHCC‐97H cells with or without NAT10 knockdown. (E) Normalized distribution of ac4C peaks on mRNA in shCtrl and shNAT10 MHCC‐97H cells. (F) Heatmaps of 125 transcripts displaying reduced ac4C peaks in shNAT10 cells. (G) Flow chart depicting RNA‐seq and Ribo‐seq. (H) Heatmaps of transcript level (RNA‐seq) and RPF abundance (Ribo‐seq). (I) Cumulative distribution of log2‐fold changes of mRNA‐normalized ribosome footprint reads (T.E) for ac4C(−) and ac4C(+) transcripts in shCtrl and shNAT10 MHCC‐97H cells (Kolmogorov‐Smirnov test or two‐tailed t‐test). (J) Polysome profiling of shCtrl and shNAT10 MHCC‐97H cells. (K) Western blot images of SUnSET assays quantifying nascent (puromycin‐labeled) peptides in shCtrl and shNAT10 MHCC‐97H cells. GAPDH used as loading control. Abbreviations: acRIP‐seq, acetylated RNA immunoprecipitation and sequencing; RNA‐seq, RNA sequencing; Ribo‐seq, ribosome profiling analyses; T.E, translation efficiency; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; RPF, ribosome protected fragment; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Modification, Knockdown, RNA Sequencing, Two Tailed Test, Western Blot, Labeling, Control, RNA Immunoprecipitation, Sequencing, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: ac4C modification enhances translation of HMGB2. (A) Schematic diagram of the strategy for pinpointing key NAT10 targets in HCC. (B) Fold changes of transcript level (RNA‐seq) and RPF abundance (Ribo‐seq) of 4 downstream targets. (C) Heatmap of RPF abundance for downstream 4 target genes and rank in ordered NAT10 targets gene list using RPF and T.E fold changes. (D) ac4C (top) and RPF (bottom) abundances on HMGB2 mRNA transcripts in shCtrl and shNAT10 MHCC‐97H cells. (E) acRIP‐qPCR analysis for indicated cells. (F) Western blot of HMGB2 protein expression in NAT10‐deficient cells. (G) HCC cells were treated with 10 µmol/L MG132 for 12 hours. (H) NAT10‐deficient or control cells transfected with pmirGLO‐HMGB2 reporter for 24 h, and HMGB2 translation efficiency defined as reporter protein production (F‐luc/R‐luc) divided by mRNA abundance . (I) Relative mRNA distribution of HMGB2 in ribosome fractions analyzed by qRT‐PCR in shCtrl and shNAT10 MHCC‐97H cells. (J) Subcutaneous xenograft model transplanted with indicated cells ( n = 6, scale bar: 1 cm). Tumor volume monitored and growth curves generated over 24 days (left panel); tumors weighed (right panel). (K) Liver orthotopic implantation models transplanted with indicated cells (scale bar: 1,000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of tumor nodules (right panel) ( n = 5). (L) Lung metastasis model transplanted with indicated cells (scale bar: 1,000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of lung metastatic nodules (right panel) ( n = 5). (M) Representative images of NAT10 and HMGB2 protein expression in HCC tissue arrays ( N = 103) by IHC (upper panel). Pearson correlation analysis between NAT10 and HMGB2 protein expression (lower panel). (N) Pearson correlation analysis between NAT10 and HMGB2 protein expression using CPTAC LIHC dataset. (E) and (H) Data are shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; CPTAC, Clinical Proteomic Tumor Analysis Consortium; qRT‐PCR, quantitative real‐time polymerase chain reaction; RNA‐seq, RNA sequencing; Ribo‐seq, ribosome profiling analyses; T.E, translation efficiency; acRIP, acetylated RNA immunoprecipitation; HMGB2, High Mobility Group Protein B2; IHC, immunohistochemistry; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Modification, RNA Sequencing, Western Blot, Expressing, Control, Transfection, Quantitative RT-PCR, Generated, Imaging, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Immunohistochemistry, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: CDS ac4C sites of HMGB2 mRNA enhance binding of eEF2. (A) Schematic of RNA affinity chromatography and MS analysis. (B) Dot blot showing ac4C levels (left) and MB (right, loading controls) in ac4C/C ssRNA probes. (C) GO biological process analysis of proteins identified in quantitative MS via the Database for Annotation, Visualization, and Integrated Discovery. (D) Venn diagram comparing proteins identified in ss‐ac4C and acFUS. (E) Volcano plot of identified proteins. Proteins significantly enriched in ss‐ac4C RNA are shown as red dots. Log2 fold change plotted on x‐axis; ‐log10 P value on y‐axis. (F) Western blot images of endogenous eEF2 and SRP68 proteins pulled down by biotin‐labeled C‐Oligos and ac4C‐Oligos from MHCC‐97H whole cell lysates. (G) RNA pull‐down assays showing dose‐dependent interaction between endogenous eEF2 and biotin‐labeled C‐Oligos (top) or ac4C‐Oligos (below). Gray signal of bands in Western blots (left) quantified by Image Master Total Lab (right). (H) RIP‐qPCR analysis of enrichment of HMGB2 mRNA on eEF2 relative to IgG in shCtrl and shNAT10 MHCC‐97H cells. (I) Western blot analysis of HMGB2 expressions in Huh7 cells co‐transfected with NAT10 CDS plasmid or eEF2 siRNA. (J) Schematic representation of acRIP‐qPCR with fragmented RNA from cells (left). ac4C in HMGB2 mRNA analyzed by acRIP‐qPCR using fragmented RNA in MHCC‐97H cells with shCtrl and shNAT10 (right). (K) Western blot analysis on HA‐HMGB2 in MHCC‐97H cells seeded in 6‐well plates and transfected with either wild‐type HMGB2 CDS or mutant1/2/3 HMGB2 CDS plasmid, along with control or NAT10‐deficient cells. (L) NAT10‐deficient or control cells transfected with either wild‐type HMGB2 CDS or mutant1/2/3 HMGB2 CDS reporter for 24 h, and the quotient of reporter protein production defines translation efficiency of HMGB2. (M) Binding of eEF2 with CDS or 5′ UTR in control or NAT10‐deficient cells analyzed by eEF2 RIP‐qPCR using fragmented RNA. (N) Western blotting showing endogenous eEF2 proteins pulled down by biotin‐labeled exon‐C RNA and exon‐ac4C RNA from MHCC‐97H whole cell lysates. (H, J, L‐N) Data are shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: MS, mass spectrometry; ac4C, N4‐acetylcytidine; MB, methylene blue; GO, Gene ontology analysis; RIP, RNA immunoprecipitates; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; eEF2, eukaryotic Elongation Factor 2; CDS, coding sequence; UTR, untranslated regions; acRIP, acetylated RNA immunoprecipitation; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Binding Assay, Affinity Chromatography, Dot Blot, Western Blot, Labeling, Transfection, Plasmid Preparation, Control, Mass Spectrometry, Sequencing, RNA Immunoprecipitation, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Characterization of the NAT10 inhibitor panobinostat. (A) Western blot and dot blot were used to detect HMGB2 expression and ac4C levels after transfection of NAT10 (WT) or G641E NAT10 mutant plasmid, respectively. (B) Flow diagram of NAT10 inhibitor screening. (C) Three‐dimensional (3D) binding model of Panobinostat in NAT10 catalytic pocket. (D) Biacore analysis revealing binding between NAT10 protein and Panobinostat. (E) Western blots for effects of Panobinostat on thermal stabilization of NAT10 protein. CETSA assayed in cell lysates. (F) Identification of direct binding between Panobinostat and NAT10 via DARTS assays. (G) Effects of various concentrations of Panobinostat on global ac4C modification in MHCC‐97H cells. Dot blot assays were conducted with total RNA. (H) Effects of various concentrations of Panobinostat on poly(A)+ RNA ac4C modification in MHCC‐97H cells. (I) Effects of Panobinostat on levels of nascent (puromycin‐labeled) peptides and expression of HMGB2. (J) Effects of Panobinostat on HMGB2 mRNA ac4C level using acRIP‐qPCR. (K) Effects of Panobinostat on HMGB2 mRNA translation efficiency. (L) RIP‐qPCR analysis was performed to determine the enrichment of HMGB2 mRNA on eEF2 relative to IgG in both control and Panobinostat‐treated cells. (J‐L) Data is shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. ** P < 0.01, *** P < 0.001. Abbreviations: WT, wild type; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; eEF2, eukaryotic Elongation Factor 2; RIP, RNA immunoprecipitates; DARTS, drug affinity responsive targets stability assay; CETSA, cellular thermal shift assay; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: Western Blot, Dot Blot, Expressing, Transfection, Mutagenesis, Plasmid Preparation, Binding Assay, Modification, Labeling, Control, Stability Assay, Thermal Shift Assay, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting N4‐acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2‐mediated HMGB2 mRNA translation

doi: 10.1002/cac2.12595

Figure Lengend Snippet: Panobinostat exhibits promising anti‐HCC efficacy in vitro and in vivo. (A) IC50 values of Panobinostat in MHCC‐97H after 48 h of treatment. (B) Effects of various concentrations of Panobinostat on colony formation abilities of MHCC‐97H cells. (C) Effects of various concentrations of Panobinostat on cell migration and invasion abilities of MHCC‐97H cells (scale bars = 100 µm). (D) Schematic diagram of generation and treatment of HCC models in mice. (E) Effects of different concentrations of Panobinostat on tumor volume and weight of subcutaneous xenograft model ( n = 8, scale bar: 1 cm). (F) Effects of various concentrations of Panobinostat on ac4C level in subcutaneous xenograft model. (G) Effects of various concentrations of Panobinostat on HMGB2 expression level in subcutaneous xenograft model. (H) Representative IHC images of HMGB2 expression in subcutaneous xenograft model (scale bar: 20 µm). (I) Liver orthotopic implantation models transplanted with indicated cells (scale bar: 1000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of tumor nodules (right panel) (0 mg, n = 8; 5‐10 mg, n = 10). (J) Lung metastasis model transplanted with indicated cells (scale bar: 1000 µm). Representative bioluminescence imaging of mice (left panel) and quantification of lung metastatic nodules (right panel) ( n = 9). (K) Graphic illustration depicts how NAT10 modulates ac4C‐mediated translation elongation to promote HCC progression. (A‐C) Data is shown as mean ± SD, n = 3. Statistical analysis: Unpaired t‐tests. ** P < 0.01, *** P < 0.001. Abbreviations: IC50, a half‐maximal inhibitory concentration; ac4C, N4‐acetylcytidine; NAT10, N‐acetyltransferase 10; HMGB2, High Mobility Group Protein B2; IHC, immunohistochemistry; SD, standard deviation.

Article Snippet: NAT10 protein (Origene, Rockville, MD, USA) was immobilized onto a CM7 chip (GE Healthcare Life Sciences, Marlborough, MA, USA) via amine coupling.

Techniques: In Vitro, In Vivo, Migration, Expressing, Imaging, Concentration Assay, Immunohistochemistry, Standard Deviation

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Structure-guided mapping and peptide validation of the p38/MK2 interaction interface. (A) The cocrystal structure of the p38/MK2 complex (PDB ID: 6TCA ). The molecular surface of p38 is shown in gray. The p38 docking groove is highlighted in yellow. MK2 is shown as green ribbons. (B) The MK2 D345-H400 docking motif bound to the p38 docking groove is colored based on its fragments tested in this study: D345-H400 is colored in green, I370–L393 in blue, and I370-L382 in red. (C) The binding curve from a fluorescence polarization assay showing high-affinity binding of FITC-labeled MK2 370–393 peptide to His-tagged p38 (EC 50 = 26.9 nM). (D) Dose–response curves from TR-FRET inhibition assays demonstrating that both MK2 370–393 and 369–382 peptides disrupt the p38/MK2 complex (IC 50 = 0.42 μM and 4.26 μM, respectively).

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Biomarker Discovery, Binding Assay, Fluorescence, Labeling, Inhibition

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Virtual screening and molecular dynamics analysis identify nilotinib as a candidate p38/MK2 PPI inhibitor. (A) Distribution of MM-GBSA binding free energies (Δ G bind ) from virtual screening of 1,040 FDA-approved drugs docked to the p38 docking groove. The p38 crystal structure (PDB ID: 6TCA ) was used for the modeling studies. Compounds with Δ G bind values more than two standard deviations below the mean (red bars, <−60.9 kcal/mol) were prioritized for further analysis. (B) Representative binding pose of carvedilol highlighting key interactions with the p38 docking groove, including hydrogen bonds with Val158, Glu160, and His126 (yellow lines), hydrophobic interactions with the nonpolar pocket defined by Ile116, Leu122, Leu130, and Val158, and a pi-pi stacking with His126 (cyan line). (C) Carvedilol’s carbazole moiety binds within the hydrophobic cleft of the p38 docking groove, which is shown as a molecular surface representation colored by electrostatic potential (red = negative, blue = positive). (D) Root-mean-square deviation (RMSD) plots from three 200 ns molecular dynamics simulations of the p38–nilotinib complex. The RMSD of protein backbone atoms is shown in aquamarine, and nilotinib in red. The PDB IDs of the p38 structures used for the modeling are indicated in the lower-left corners. (E) The representative binding pose of nilotinib obtained after 200 ns MD simulation (a final snapshot of one of the MDs), highlighting pi-pi and H-bond interactions with His126, the H-bonding with Glu160, and multiple water-bridged contacts that stabilize ligand orientation within the groove. (F) Structural overlay of the nilotinib–p38 complex with the p38/MK2 cocrystal structure, illustrating displacement of key MK2 anchoring residues Ile372 and Ile375 by nilotinib. P38 is shown as green ribbons, the p38 docking groove as gray molecular surface, and MK2 as red ribbons.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Validation of nilotinib as a p38/MK2 PPI Inhibitor. (A) Thermal shift assay (TSA) showing dose-dependent stabilization of recombinant His-tagged p38 by nilotinib (Δ T max = 8.22 °C), consistent with direct binding. (B) TSA profile for SR318, a type II ATP-competitive p38 inhibitor, used as a positive control (Δ T max = 13.47 °C). (C) Nilotinib competes with His-MK2 346–400 fragment for VF-p38 in a cell lysate-based TR-FRET assay. (D) Quantitative qRT-PCR analysis showing that nilotinib significantly ( p -value <0.05) suppresses LPS-induced TNF-α, IL-6, and IL-1β expression in HMC3 microglial cells. P38 inhibitors SR318 and VX-745 were used as positive controls. (E) Nilotinib disrupts the endogenous p38/MK2 complex in HMC3 cells, as shown by coimmunoprecipitation, correlating with cytokine suppression. (F) qRT-PCR analysis showing that nilotinib suppresses LPS/IFNγ-induced TNF-α expression in the human iPSC-derived microglia (iMGL). (G) TR-FRET assay with recombinant p38 and MK2 proteins purified from E. coli demonstrated direct inhibition of the complex by nilotinib (IC 50 = 2.2 μM). In contrast, ATP-site inhibitors VX-745 and SR318 failed to disrupt the interaction, supporting a non-ATP-competitive mechanism for nilotinib activity. (H) Nilotinib demonstrates a weak inhibition of p38/ATF2 PPI (IC 50 > 30 μM, maximal inhibition ∼ 37%) in a TR-FRET assay with recombinant purified His-p38 and GST-ATF2. The inhibition of His-p38/GST-MK2 PPI by nilotinib was monitored in parallel.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Biomarker Discovery, Thermal Shift Assay, Recombinant, Binding Assay, Positive Control, Quantitative RT-PCR, Expressing, Derivative Assay, Purification, Inhibition, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Chemical structures of nilotinib and ten analogs evaluated for p38/MK2 PPI inhibition using a TR-FRET assay with recombinant purified proteins. IC 50 values are shown for compounds exhibiting measurable activity; compounds with less than 50% inhibition at 30 μM are indicated as not determined (N.D.).

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Inhibition, Recombinant, Purification, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Field-based QSAR maps illustrating physicochemical features of nilotinib analogs associated with p38/MK2 PPI inhibition. (A) Compounds 1–6 (white) and 7–10 (orange) docked into the p38 binding groove. The molecular surface of the binding groove is colored based on the electrostatic potential, ranging from the most positive (blue) to the most negative (red) charge. (B) Steric field map showing regions where steric bulk is favorable (green). The pyridine–pyrimidine system is positioned within favorable steric zones, supporting its critical role in activity. (C) Hydrophobic field map with yellow-green and gray surfaces representing positive and negative hydrophobic contributions, respectively. (D) Electrostatic field map colored by potential (red - negative, blue - positive). (E) Hydrogen bond acceptor field map. Red contours indicate favorable contributions of H-bond acceptors, while the magenta contour indicates unfavorable contributions of H-bond acceptors. (F) Hydrogen bond donor field map. The blue-violet contour indicates the region favorable for H-bond donors. The cyan field map indicates the area unfavorable for the H-bond donors.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Inhibition, Binding Assay, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Development of a lysate-based TR-FRET platform for high-throughput screening of p38/MK2 PPI inhibitors. (A) The preferential binding of MK2 to p38α and p38β isoforms was determined by Flag-immunoprecipitation in HEK293T cells. (B) Isoform selectivity of MK2 binding was validated by TR-FRET using lysates coexpressing GST-tagged MK2 and Venus-Flag (VF)-tagged p38 isoforms. Robust signal was observed for p38α and p38β, with negligible interaction detected for p38γ and p38δ. (C) TR-FRET assay shows stable signal over 48 h postantibody addition, indicating excellent temporal stability. (D) The platform tolerates up to 10% DMSO without signal degradation, supporting its suitability for screening applications. (E) Pilot screen of 2036 compounds from the Emory Enriched Library (EEL) in 1536-well format identified 48 compounds that inhibited the p38/MK2 interaction by ≥ 50% relative to vehicle control. Gray dots indicate fluorescence assay-interfering compounds.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: High Throughput Screening Assay, Binding Assay, Immunoprecipitation, Control, Fluorescence

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: α 1 -Adrenergic antagonists disrupt the p38/MK2 interface and suppress cytokine production in microglial cells. (A) Chemical structures of doxazosin, terazosin, and alfuzosin, three α 1 -adrenergic receptor antagonists identified from the high-throughput screen. (B) Dose–response TR-FRET assays using recombinant purified p38 and MK2 proteins demonstrate that all three compounds inhibit the p38/MK2 protein–protein interaction, with IC 50 values of 4.4 μM (doxazosin), 6.2 μM (terazosin), and 6.9 μM (alfuzosin). (C) The compound activity was confirmed in a cell lysate-based TR-FRET format, showing a moderate reduction in potency relative to the recombinant protein assay. (D) In a complementary TR-FRET assay using HEK293T lysates coexpressing VF-tagged p8 and a His-tagged MK2 346–400 docking peptide, all three α 1 -antagonists and nilotinib dose-dependently disrupted peptide binding to p38, consistent with direct competition at the docking interface. (E) qRT-PCR analysis in HMC3 microglial cells shows that all three compounds significantly ( p -values <0.05) suppressed LPS-induced expression of TNF-α, IL-6, and IL-1β, similarly to known p38 inhibitors SR318 and VX745, demonstrating effective functional inhibition of p38/MK2 signaling in a disease-relevant context.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: High Throughput Screening Assay, Recombinant, Purification, Activity Assay, Binding Assay, Quantitative RT-PCR, Expressing, Functional Assay, Inhibition

Journal: Journal of Medicinal Chemistry

Article Title: Nonpeptidic Oxazole-Based Prolyl Oligopeptidase Ligands with Disease-Modifying Effects on α-Synuclein Mouse Models of Parkinson’s Disease

doi: 10.1021/acs.jmedchem.3c00235

Figure Lengend Snippet: Cellular thermal shift assay (CETSA) shows target engagement for 10 μM HUP-55 on PREP in HEK-293 cells (A). HUP-55 at 1 or 10 μM concentration does not inhibit close relative enzymes FAP, DPP4, DPP9, or DPP2 (B). In HEK-293 cells, HUP-55 decreases αSyn dimerization (C), but when PREP is not expressed (PREP knock-out (KO) HEK-293 cells), no such effect is seen (D). A similar effect was seen in autophagy when assayed with LC3BII protein levels in HEK-293 (E) and PREP-KO HEK-293 cells (F), indicating a PREP-mediated effect. Data are presented as mean + SEM. **, p < 0.01; student’s t -test.

Article Snippet: DPP2: Recombinant human DPP2 was purchased from R&D Systems (#3438-SE-010).

Techniques: Thermal Shift Assay, Drug discovery, Concentration Assay, Knock-Out

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet: Cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) assay for evaluation of drug-PBX1 protein interaction (A) Western blot analysis of cellular thermal shift assays (CETSAs). SKOV3 cells stably expressing the PBX1-V5 protein were treated with 50 μM of each compound for 1 h and subsequently heated at 37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, or 58°C for 3 min. Soluble PBX1 protein bound to the test compound was visualized by western blot using an anti-V5 antibody. The detailed protocol is provided in . (B) Quantification of CETSA melt curves based on the PBX1-V5 band intensities shown in (A). Data are normalized to band intensities obtained at 37°C. (C) Bar graph showing the results of CETSAs measured at 52°C and 58°C. Data represent means ± SD. (D) SPR sensogram showing purified PBX1 protein (PBX1-DBD) binding to immobilized double-stranded DNA probes containing PBX1 binding motif (left) or without PBX1 binding motif (right) at six concentrations, ranging from 3.125 to 100 μM in triplicate. (E) Test efficacy of small molecular inhibitor T417 or its analog in interfering with the PBX1 protein-DNA probes interaction. SPR sensogram shows PBX1 protein (PBX1-DBD) (red), PBX1 protein + inhibitor (blue), and inhibitor (green) binding to the immobilized DNA probes containing the PBX1 binding motif. Each experiment was performed in duplicate. (F) T417 competition of PBX1 protein binding to immobilized PBX1 consensus DNA at nine concentrations ranging from 0 to 80 μM in duplicate. Data are normalized to binding in the absence of T417 inhibitor. The IC 50 derived is 5 μM.

Article Snippet: Cell lines, including 293T, OVCAR3, SKOV3, and A2780, were purchased from ATCC (Rockville, MD).

Techniques: Thermal Shift Assay, SPR Assay, Western Blot, Stable Transfection, Expressing, Purification, Binding Assay, Protein Binding, Derivative Assay

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet: PBX inhibitor selectively inhibits carboplatin-resistant tumor cells (A) Western blot analysis of PBX1 expression by OVCAR3 and SKOV3 naive, carboplatin-resistant (CR) and paclitaxel-resistant (TR) cells. The inhibitor was incubated for 48 h, and cell viability was measured using a CellTiter blue assay. (B) Cell viability of SKOV3 and OVCAR3 naive, CR, and TR cells under a serial concentration of T417. T417 was incubated for 48 h, and cell viability was measured using a CellTiter blue assay. (C) Spherogenic capacity of OVCAR3-CR and SKOV3-CR cells treated with T417. (D) SKOV3 and OVCAR3 naive and CR cells were treated with a serial concentration of carboplatin combined with or without T417. Cell viability was measured using a CellTiter-Glo 3D assay and was normalized to the viability of cells in the absence of an inhibitor.

Article Snippet: Cell lines, including 293T, OVCAR3, SKOV3, and A2780, were purchased from ATCC (Rockville, MD).

Techniques: Western Blot, Expressing, Incubation, CtB Assay, Concentration Assay

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet: PBX1 inhibitor suppresses tumor growth in xenograft models of carboplatin-resistant cancer models (A) Western blot analysis of PBX1 expression in SKOV3 parental, SKOV3 CR, and A2780 cells. (B) The tumor volume of the SKOV3 parental tumor model treated with T417 (5 mg/kg/injection) or vehicle (1% DMSO). Tumor size was measured twice per week by a caliper. (C) At the end of the treatment in (B), SKOV3 parental tumors were excised and mRNA was extracted for measuring PBX1-downstream target genes, MEOX1 and BCL6, by qRT-PCR. (D and E) Tumor volume and expression of PBX1-downstream target genes in an SKOV3 CR tumor model treated with vehicle, carboplatin (30 mg/kg/injection), T417 (5 mg/kg/injection), or the combination of carboplatin and T417. In (E), the relative expression levels of MEOX1 and BCL6 were measured by qRT-PCR. (F) Tumor volume and tumor weight of the A2780 xenograft tumor models treated with vehicle, carboplatin (30 mg/kg/injection), T417 (5 mg/kg/injection), or the combination of carboplatin and T417. Tumor growth was monitored as in (B and D). (G) Expression of PBX1-downstream target genes in A2780 xenograft tumors was determined as in (G).

Article Snippet: Cell lines, including 293T, OVCAR3, SKOV3, and A2780, were purchased from ATCC (Rockville, MD).

Techniques: Western Blot, Expressing, Injection, Quantitative RT-PCR

Journal: iScience

Article Title: Development of small molecule inhibitors targeting PBX1 transcription signaling as a novel cancer therapeutic strategy

doi: 10.1016/j.isci.2021.103297

Figure Lengend Snippet:

Article Snippet: Cell lines, including 293T, OVCAR3, SKOV3, and A2780, were purchased from ATCC (Rockville, MD).

Techniques: Recombinant, Western Blot, Immunoprecipitation, SYBR Green Assay, Staining, Stripping, Extraction, Luciferase, Purification, Transfection, cDNA Synthesis, Viability Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation decreases methyltransferase activity and intrinsic protein stability of SETD2 catalytic core in vitro . A , upper panel : schematic representation of the SETD2 domains. The SETD2 L1609P mutation is located in the SET domain within the SETD2 catalytic core (composed of the AWS, SET, and post-SET domains). Lower left panel : Structural representation of the SETD2 active site (PDB entry: 5JJY ) with a zoomed-in view of the substrate (H3K36M peptide) and cofactor (SAH) binding sites. Lower right panel : Sequence alignment of residues 1603 to 1619 of the SET domain of human SETD2 with the equivalent sequences of human G9A, EZH2, NSD1, NSD2, SETD8, MLL1, MLL2, SETD8, ASH1 (sequence retrieved from the UniProt database). Conserved residues are highlighted in blue . The secondary structure of the SETD2 residues (deduced from PDB entry: 5JJY ) is shown above the alignment. The SETD2 residue L1609 and the equivalent residues in the other SET domain-containing enzymes are highlighted in orange . B , in vitro methylation of recombinant histone H3, core histones (purified from HEK293T SETD2-KO cells) or recombinant nucleosomes. SETD2-dependent H3K36me3 methylation was detected using an anti-H3K36me3 antibody. Ponceau Red staining of histones is shown. The purified catalytic core of SETD2 WT and SETD2 L1609P mutant used in the assays were detected using an anti-6xHis-tag antibody. C , SETD2 mono-methylation, dimethylation, or trimethylation activities were determined by UFLC assays using H3K36 fluorescent peptides as previously described ( , ). Bar graphs and error bars represent the mean and SD of three independent experiments. D , automethylation of SETD2 and methylation of α-tubulin detected by autoradiography using 3 H-SAM. Coomassie Blue staining was used as loading control. E , determination of the intrinsic protein stability of SETD2 WT or SETD2 L1609P by thermal shift assay (TSA). Left panel : T m values were determined by the minimum of the first derivative of the fluorescence emission as a function of temperature (dFluo/dT). Right panel : Bar graphs and error bars represent the mean and SD of nine experiments. SETD2, SET-domain containing protein 2; UFLC, ultrafast liquid chromatography.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Activity Assay, In Vitro, Binding Assay, Sequencing, Residue, Methylation, Recombinant, Purification, Staining, Autoradiography, Control, Thermal Shift Assay, Fluorescence, Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation results in low levels of the H3K36me3 mark and in low expression of SETD2 in CRISPR/Cas9-engineered HEK293T cells and in transfected HEK293T-SETD2 KO cells . A , endogenous H3K36me3 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : the H3K36me3 mark was detected by immunofluorescence using an anti-H3K36me3 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with 10 μm scale bars. Right panel : Histones from CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant were extracted and H3K36me3 levels were determined by Western blotting using a an anti-H3K36me3 antibody. Ponceau Red staining of extracted histones is shown. B , endogenous SETD2 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : Cells were fixed and SETD2 was detected using an anti-SETD2 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with scale bars of 10 μm. Right panel : SETD2 was detected in cell extracts by Western blot using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. C , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 L1609P were transfected with GFP-SETD2 WT or GFP-SETD2 L1609P plasmids. Nontransfected CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were used as controls. Ectopic GFP-SETD2 expression and H3K36me3 mark levels were detected by Western blot using anti-GFP or anti-H3K36me3 antibodies, respectively. Ponceau Red staining of cellular histones or extracts on membranes are shown. D , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were treated with MG132 or DMSO. Endogenous SETD2 WT and SETD2 L1609P expression levels were detected by Western blotting using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Expressing, CRISPR, Transfection, Immunofluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Overall structure of the ternary complex of SETD2 L1609P mutant bound to H3K36M peptide and SAM cofactor . A , left panel : cartoon representation of SETD2 WT (PDB: 5JJY ) ( cyan ) bound to H3K36M peptide ( orange ) and the SAH cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 L1609 and H3M36 residues are represented by yellow and orange sticks , respectively. The close-up view shows the region around residue L1609 with the H3K36M peptide (residues 29–42, orange ) and the SAH cofactor ( black sticks ). Zinc atoms are shown in gray . Right panel : cartoon representation of the SETD2 L1609P mutant (PDB: 8RZU ) ( salmon ) bound to the H3K36M peptide ( green ) and the SAM cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 P1609 and H3M36 residues are shown as yellow and green sticks , respectively. The close-up view shows the region around the residue P1609 with the H3K36M peptide (residues 29–39, green ) and the SAM cofactor ( black sticks ). B , left panel : cartoon representation of the characteristic triangular shape of the SET domain formed by 3 β-sheets (β1-β2; β3-β8-β7; β4-β6-β5 strands) of SETD2 WT in complex with the H3K36M peptide (residues 29–42 in orange) (PDB: 5JJY ). The β-sheet composed of β4-β6-β5 strands is boxed and the SETD2 L1609 residue is shown in yellow . Right panel : cartoon representation of the triangular β-sheet structure of the SET domain of the SETD2 L1609P mutant ( salmon ) in complex with the H3K36M peptide (residues 29–39, green ) (PDB: 8RZU ). The β5-strand in SETD2 WT adopts a loop conformation in the structure of the SETD2 L1609P mutant ( boxed ). The P1609 residue in mutant SETD2 is shown in yellow . SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Effects of the SETD2 L1609P mutation on the conformations of neighboring residues of SETD2 and the H3K36M peptide. A , the left panel shows a cartoon overlay of the β5-β6 hairpin of SETD2 WT (PDB: 5JJY ) ( cyan ) and SETD2 L1609P mutant ( salmon ) structures. The H3K36M peptide is shown in orange and green for SETD2 WT and SETD2 L1609P, respectively. The side chains of residues L1609 and P1609 residues are shown as sticks ( yellow CPK). The middle panel shows a close-up view of the hairpin residues (1609–1613) of SETD2 WT ( cyan ) and SETD2 L1609P ( salmon ). The side chains are shown in CPK sticks . The right panel shows the β5-β6 hairpin residues of SETD2 WT ( top ) and SETD2 L1609P ( bottom ) in sticks . Dashes represent the distance between Cα of residues K1610 and E1613 residues. B , conformational remodeling of residues K1610 and K1639 of SETD2 and residue K37 of H3 induced by the L1609P mutation. Left panel shows residues SETD2 L1609 ( yellow ), K1610 (cyan), K1639 ( cyan ), and H3K37 ( orange ) in spheres and sticks in the SETD2 WT structure (PDB: 5JJY ). Middle panel shows residues SETD2 P1609 ( yellow ), K1610 ( salmon ), K1639 ( salmon ), and H3K37 ( green ) in spheres and sticks in the SETD2 L1609P structure. The right panel shows residues P1609 ( yellow ) and K1610 ( salmon ) from the SETD2 L1609P structure and residues K1639 ( cyan ) and H3K37 ( orange ) from the SETD2 WT structure. Steric clashes between side chains are shown in boxes . The orientations are the same in all three panels and were obtained by superimposing the SETD2 WT and L1609P main chains. C , surface representation of the SETD2 substrate-binding region. H3K36M peptides are shown as sticks. The left panel shows the SETD2 WT structure (PDB: 5JJY ) in light cyan . The SETD2 L1609 residue is shown in yellow . The SETD2 K1610 and K1639 residues are shown in blue . H3K36M peptide residues diffracting in both WT and L1609P structures (residues A29–H39) are shown in green . H3K36M peptide residues observed only in the SETD2 WT structure (residues R40-R42) are shown in transparent orange . The right panel shows the SETD2 L1609P structure in light pink . The SETD2 P1609 residue is shown in yellow . The K1610 and K1639 residues are shown in purple . H3K36M peptide residues observed in the SETD2 L1609P structure (A29–H39) are shown in green . SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Details of H3K36M peptide recognition by SETD2 L1609P mutant . A , the left panel shows a clipped surface representation of the SETD2 WT-H3K36M peptide complex (PDB: 5JJY ). Peptide residues (residues A29–R42) are represented by sticks . The right panel shows a clipped surface representation of the SETD2 L1609P-H3K36M peptide complex. Peptide residues (A29–H39) are represented by sticks . The structures of the SETD2-H3K36M peptide complexes are shown in the same orientation after superimposition of the main chains. B , upper panel : Structural alignment of H3K36M peptides (residues A29–H39) in SETD2 WT (PDB: 5JJY ) ( orange ) and SETD2 L1609P ( green ) structures. Lower panel : Differences between SETD2-H3K36M peptide interactions in SETD2 WT and SETD2 L1609P complexes. Residue interactions across the binding interface of SETD2 WT or SETD2 L1609P mutant with H3K36M peptide were determined using LIGPLOT . Residues are represented by sticks . Residues involved in SETD2-H3K36M peptide interactions (nonbonded and hydrogen bonds) are represented by sticks and spheres . Dashes represent hydrogen bond. The lower left panel shows the SETD2 WT ( cyan )-H3M36 ( orange ) interacting residues that are specific for the SETD2 WT complex and not present in the SETD2 L1609P complex. These interactions are listed in a table ( bottom left ). The lower right panel shows SETD2 L1609P ( salmon )-H3K36M ( green ) peptide interacting residues that are specific for the SETD2 L1609P complex and not present in the SETD2 WT complex. These interactions are listed in a table ( bottom right ). SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . Effects of FGF1 ΔHBS -FGF21 C-tail on blood glucose levels and insulin sensitivity in db/db mice (a) Changes in blood glucose levels of db/db mice following an acute intraperitoneal injection of FGF1 ΔHBS -FGF21 C-tail , FGF21 WT or FGF1 ΔHBS (0.5 mg/kg body weight) over the course of 24 h; db/db mice treated with buffer alone (PBS) and their littermates ( db/m ) served as controls. Data are presented as mean +/- SEM ( n = 5). * p <0.05, ** p <0.01, *** p <0.001 vs db/db ; # p <0.05, ## p <0.01, ### p <0.001 vs FGF21 WT treatment, unpaired t -test. (b–d) Fed-state blood glucose levels (b), body weight (c) and food intake (d) of db/db mice over the course of 24 days of daily subcutaneous administration of FGF1 ΔHBS -FGF21 C-tail , FGF21 WT or FGF1 ΔHBS (0.5 mg/kg body weight). Data are presented as mean +/- SEM ( n = 6). * p <0.05, *** p <0.001 vs db/db ; ### p <0.001 vs FGF21 WT treatment, unpaired t -test. (e) Serum insulin levels of db/db mice after a 24-day course of treatment with FGF1 ΔHBS -FGF21 C-tail , FGF21 WT or FGF1 ΔHBS administered as in (b). Data are presented as mean +/- SEM ( n = 6). ** p <0.01, *** p <0.001 vs db/db ; ## p <0.01 vs FGF21 WT treatment, unpaired t -test. (f,g) Glucose tolerance test in db/db mice performed 12 h after the final injection (on day 24) of sequential administration with FGF1 ΔHBS -FGF21 C-tail , FGF21 WT or FGF1 ΔHBS as detailed in (b). Changes in blood glucose levels (e) and integrated area under the curve (AUC) (f) are shown. Data are presented as mean +/- SEM ( n = 6). * p <0.05, *** p <0.001 vs db/db ; ### p <0.001 vs FGF21 WT treatment, unpaired t -test.

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Injection

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . The atypical trefoil core domain of endocrine FGFs imparts thermal instability and a weak receptor binding affinity (a) Structural comparison of paracrine and endocrine FGFs highlighting major differences in their HS binding domains. The view is from the top looking down onto the β–trefoil core. An overlay of Cα traces of four paracrine FGFs [FGF1 (PDB: 2AFG), FGF2 (PDB: 4OEE), FGF4 (PDB: 1IJT), FGF9 (PDB: 1IHK)] and three endocrine FGFs [FGF19 (PDB: 2P23), FGF23 (PDB: 2P39)] is shown. For FGF21, a homology model was used because an experimentally derived crystal structure is lacking. The FGF21 model was generated using an automated SWISS-MODEL homology-modeling pipeline (www. swissmodel. expasy. org) on the basis of the structure of its subfamily member FGF19 (PDB: 2P23). (b) Close-up view of the major HS binding site between β10 and β12 strands of FGF1 (used as a representative of paracrine FGFs) and FGF21 or FGF23. Note that endocrine FGFs do not have an equivalent β11 strand and are therefore atypical. (c) Superimposition of the atypical β-trefoil core of endocrine FGFs (FGF19/FGF21/FGF23) onto the canonical β-trefoil core of FGF1/FGF2/FGF4/FGF9. Locations of glycine and threonine residues of the GXXXXGXX (T/S) motif conserved only in paracrine ligands (FGF1, FGF2, FGF4 and FGF9) are highlighted by red dots. In a-c, FGF19, FGF21, FGF23, FGF1, FGF2, FGF4 and FGF9 are indicated in pink, purple, gray, green, light blue, red and orange, respectively. (d) Comparison of melting temperatures (T m ) of paracrine FGFs (FGF1/FGF2/FGF4/FGF9) and endocrine FGFs (FGF19/FGF21/FGF23) obtained by a fluorescence dye-based thermal shift assay. T m values from three independent measurements are presented as mean ± SEM. (e) Crystal structure of FGF1-FGFR1c (PDB: 3OJV). FGFR1c (comprising D2 and D3 domains) and FGF1 are represented as a cyan molecular surface and a green cartoon, respectively. (f) An expanded view of the region boxed in (E) showing contacts between the hydrophobic patch of FGF1 and the D3 domain of FGFR1c. Selected residues in FGF1 (S62/E64/S65/V66/Y70/Y79/P94, labeled in red or green) and the FGFR1 D3 domain (labeled in blue) are represented as sticks. The β4 and β5 strands of FGF1 are labeled. Note that FGF1 residues labeled in red are not conserved between FGF21 and FGF1 (see panel g). A hydrogen bond between E64 in FGF1 and H286 in FGFR1 is depicted as a dashed line. Oxygen and nitrogen atoms are shown in red and blue, respectively; NT, CT denote the location of the FGF1 N- and C-termini. (g) Structure-based sequence alignment of FGF1 WT (aa 59–73) and FGF21 WT (aa 54–68). Secondary structure elements are indicated on top of the sequence alignment. The five residues in FGF21 WT that were mutated so as to correspond to those in FGF1 WT (thus generating FGF21 mut ) are highlighted in red. (h-j) Representative SPR sensorgrams of binding interactions between FGF1 WT (h), FGF21 WT (i) and FGF21 mut (j) with FGFR1c. FGF1 WT , FGF21 WT and FGF21 mut at the indicated concentrations were passed over CM5 chips containing the immobilized FGFR1c ligand-binding domain. In (h), K d values from three independent measurements are presented as mean ± SEM.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Comparison, Derivative Assay, Generated, Fluorescence, Thermal Shift Assay, Labeling, Sequencing, Ligand Binding Assay

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . Design and characterization of a thermally stable, high FGFR binding affinity FGF1 ΔHBS -FGF21 C-tail chimera (a) Core and C-terminal tail regions of endocrine FGFs play independent roles in ternary complex formation. Image at center shows the crystal structure of the FGF23-FGFR1c-αKlotho complex (PDB:5W21); the core and C-tail of endocrine FGF are delineated within dashed black and blue boxes, respectively. In image at left, αKlotho has been removed to visualize the fact that the binding of endocrine FGFs to FGFR is mediated exclusively by the core region. In image at right, the receptor has been removed to illustrate the fact that the unique C-tail of endocrine FGF is specialized for αKlotho co-receptor binding. Endocrine FGF ligand, FGFR1c and Klotho are coloured in orange, cyan and green, respectively. (b) Left: crystal structure of the FGF1-FGFR1c complex (PDB ID: 1EVT) with a modeled bound HS oligosaccharide; an expanded view is shown at top right. Modeling of HS is based on the crystal structure of the FGF2-FGFR1c-HS ternary complex (PDB ID: 1FQ9). FGF1 WT and FGFR1c are coloured green and cyan, respectively; the HS oligosaccharide carbon backbone is shown in grey sticks. Side chains of three lysine residues of FGF1 predicted to make major contacts with HS are shown as sticks; black dashed lines denote hydrogen bonds. Lower right: Linear representations of human FGF1 (Met1-Asp155) (shown in green), FGF21 (His29-Ser209) (core and C-tail shown in yellow and orange, respectively) and the chimeric molecule FGF1 ΔHBS -FGF21 C-tail composed of the HS-binding defective endocrinized FGF1 and the C-terminal tail of FGF21 (Gly169-Ser209). (c-f) Representative SPR sensorgrams of HS binding abilities of FGF1 WT , FGF21 WT and FGF1 ΔHBS -FGF21 C-tail (c), βKlotho binding ability of FGF1 ΔHBS (d), FGF21 WT (e) and FGF1 ΔHBS -FGF21 C-tail (f). (g) T m values of FGF21 WT and FGF1 ΔHBS -FGF21 C-tail obtained by fluorescence dye-based thermal shift assays. (h) Pharmacokinetics of FGF21 WT and FGF1 ΔHBS -FGF21 C-tail following injection into rats (Log C-T). (i) Half-lives of FGF21 WT and FGF1 ΔHBS -FGF21 C-tail in vivo based on the data shown in (h). Data are shown as mean +/- SEM ( n = 6). *** p <0.001 vs FGF21 WT unpaired t -test. (j) Representative SPR sensorgrams of FGFR1c binding ability of FGF1 ΔHBS -FGF21 C-tail . In panels c-g, j, the various proteins at the indicated concentrations were passed over the sensor chip containing the indicated immobilized molecule shown in the panels.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Fluorescence, Drug discovery, Injection, In Vivo

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . FGF1 ΔHBS -FGF21 C-tail is impaired in HS-dependent signalling (a) SEC-MALS analysis of a 1:1:1 stoichiometric mixtures of FGF1, FGFR1c and HS (green) and FGF1 ΔHBS -FGF21 C-tail , FGFR1c and HS (red). Y axes show scales representing molecular mass (left) and A 280 (right). Molecular masses of the protein components used were FGF1 WT (17.4 kDa), FGF1 ΔHBS -FGF21 C-tail (21.0 kDa), FGFR1c ligand-binding domain (25.4 kDa) and HS (3.0 kDa). For each complex, the peak region used for molecular mass determination is demarcated, together with a horizontal dashed line showing the corresponding molecular mass. (b) Western blots showing dose-dependent phosphorylation of FGFR1 and ERK1/2 in NIH-3T3 fibroblasts induced by FGF1 WT and FGF1 ΔHBS -FGF21 C-tail at the concentrations shown. (c) NIH-3T3 fibroblast cell proliferation in response to FGF1 WT (green), FGF1 ΔHBS -FGF21 C-tail (red), or a PBS control (black) using an MTT assay (see Methods). Data from three independent measurements are presented as mean +/- SEM. *** P <0.001 vs FGF1 WT , unpaired t -test. (d) Liver tissue from C57BL/6 J mice chronically treated by intravenous injection with FGF1 WT , FGF1 ΔHBS -FGF21 C-tail (2.0 mg/kg body weight per day) or a PBS control (vehicle) were immunostained with an anti-Ki67 antibody. Scale bar, 100 µm. (e) Quantitation of immunohistochemical staining data shown in (D) using ImageJ software. Data are presented as mean +/- SEM ( n = 4). ### p <0.001 vs vehicle; *** p <0.001 vs FGF1 WT , unpaired t -test. (f) Western blot analysis of Ki67 level in livers from mice chronically treated with FGF1 WT , FGF1 ΔHBS -FGF21 C-tail , or a PBS control (vehicle). An actin loading control is shown below. ( g ) Quantification of western blot results shown in (f) using Image J software. Data are presented as mean +/- SEM ( n = 4). ### p <0.001 vs vehicle; *** p <0.001 vs FGF1 WT , unpaired t -test.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Ligand Binding Assay, Western Blot, Phospho-proteomics, Control, MTT Assay, Injection, Quantitation Assay, Immunohistochemical staining, Staining, Software

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . βKlotho promotes signalling by the FGF1 ΔHBS -FGF21 C-tail chimera (a-d) Western blot analyses showing dose-dependent phosphorylation of ERK1/2 induced by FGF21 WT and FGF1 ΔHBS -FGF21 C-tail in BaF3 cells expressing FGFR1c alone (a), BaF3 cells co-expressing FGFR1c and βKlotho (b), and dose-dependent phosphorylation of FGFR1c and ERK1/2 induced by FGF21 WT and FGF1 ΔHBS -FGF21 C-tail in undifferentiated (c) and differentiated (d) 3T3L1 adipocytes. (e) Relative values of ERK1/2 phosphorylation in undifferentiated and differentiated 3T3L1 adipocytes induced by a range of concentrations of FGF21 WT and FGF1 ΔHBS -FGF21 C-tail . n = 3/concentration group. (f) Left hand panels: Distribution of Alexa 647 Fluor-labeled FGF1 ΔHBS -FGF21 C-tail, FGF21 WT and FGF1 ΔHBS proteins in C57BL/6 J mice at 0, 15 and 30 mins post-intraperitoneal injection (0.5 mg/kg body weight). Right hand panels: Ex vivo fluorescence imaging of the indicated organs harvested from the same mice. Injection with fluorescent dye alone was used as a control (Ctrl). Vertical scale at right shows colour correspondence with fluorescent intensity. (g) Western blot analyses of ERK1/2 phosphorylation in lysates prepared from WAT, liver, pancreas, muscle, kidney, heart, lung, spleen, small intestine and stomach from C57BL/6 J mice 30 min after subcutaneous injection with PBS (vehicle), FGF1 ΔHBS -FGF21 C-tail , FGF1 ΔHBS or FGF21 WT (0.5 mg/kg body weight). For each tissue, an ERK1/2 antibody was used as a loading control. ( h ) Quantitation of western blot data from (g) using Image J software. Data are presented as mean +/- SEM ( n = 6). *** p <0.001, FGF1 ΔHBS , FGF21 WT and/or FGF1 ΔHBS -FGF21 C-tail vs vehicle; ## p <0.01, ### p <0.001, FGF1 ΔHBS vs FGF1 ΔHBS -FGF21 C-tail ; && p <0.01, &&& p <0.001, FGF21 WT vs FGF1 ΔHBS -FGF21 C-tail , unpaired t -test.

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Phospho-proteomics, Expressing, Concentration Assay, Labeling, Injection, Ex Vivo, Fluorescence, Imaging, Control, Quantitation Assay, Software

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . Chronic Effects of FGF1 ΔHBS -FGF21 C-tail on adipose remodeling in db/db mice Adipose tissues were isolated from db/db mice treated with FGF1 ΔHBS -FGF21 C-tail or FGF21 for 24 days (0.5 mg/kg body weight); corresponding tissues from db/db mice treated with buffer alone (PBS) and their littermates ( db/m ) served as controls. (a) Epididymal fat weight of db/db mice treated with FGF1 ΔHBS -FGF21 C-tail or FGF21 WT . Data are presented as mean +/- SEM ( n = 6). *** p <0.001 vs db/db ; ## p <0.01 vs FGF21 WT treatment, unpaired t -test. (b,c) Images of WAT (b) and brown adipose tissue (BAT) (c) sections stained with hematoxylin and eosin (H&E). Data are representative of 6 mice from each group. Scale bar, 100 µm. Right hand panels: relative adipocyte sizes calculated from H&E stained sections (Relative sizes=unit area/ the cell number per unit area). Data are presented as mean +/- SEM ( n = 6). ** p <0.01, *** p <0.01 vs db/db ; # p <0.05, ## p <0.01 vs FGF21 WT treatment, unpaired t -test. (d,e) Western blot analysis for Epi-WAT AKT phosphorylation and its quantitation by image J. Data are presented as mean +/- SEM ( n = 6). * p <0.05, *** p <0.001 vs db/db ; ## p <0.01 vs FGF21 WT treatment, unpaired t -test. (f,g) Enhanced translocation of GLUT1 in Epi-WAT as measured by immunofluorescence staining (f) and quantified by image J (g). Data are representative of 6 mice from each group; *** p <0.001 vs db/db ; ### p <0.001 vs FGF21 WT treatment, unpaired t -test . Scale bar, 100 μm. (h,i) Real-time quantitative PCR analysis of mRNAs encoding PGC-1α (h) and UCP-1 (i) in subcutaneous adipose tissue. Data are presented as mean +/- SEM ( n = 6). *** p <0.001 vs db/db ; ## p <0.01, ### p <0.001 vs FGF21 WT treatment, unpaired t -test.

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Isolation, Staining, Western Blot, Phospho-proteomics, Quantitation Assay, Translocation Assay, Immunofluorescence, Real-time Polymerase Chain Reaction

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . Effects of FGF1 ΔHBS -FGF21 C-tail on hepatic steatosis and lipid metabolism in db/db mice The db / db mice were chronically treated by daily subcutaneous injection of PBS alone (as a control) or FGF1 ΔHBS -FGF21 C-tail or FGF21 WT proteins (0.5 mg/kg body weight per day) over the course of 24 days. (a) Changes in liver mass of db/db mice. Data are presented as mean +/- SEM ( n = 6). * p <0.05 vs db/db, # p <0.05 vs FGF21 WT treatment, unpaired t -test. (b) Images of liver tissue sections stained with hematoxylin and eosin (H&E) (upper panel) or Oil Red O (lower panel). Red dots in the Oil Red O stained tissue sections are indicative of micro- and macro-vesicular steatosis; note the greater improvement following administration of the chimera compared to administration of FGF21 WT . Data are representative of 6 mice per group. Scale bar, 100 µm. ( c–e ) Triglyceride (TG) (c), alanine transaminase (ALT) (d) and aspartate transaminase (AST) (e) levels in liver extracts (see Methods). Data are presented as mean +/- SEM ( n = 6). * p <0.05, ** p <0.01, *** p <0.001 vs db/db; # p <0.05 vs FGF21 WT treatment, unpaired t -test. (f) Western blot analyses of liver tissues of db/db mice following 24-day chronic treatment with FGF1 ΔHBS -FGF21 C-tail . Note the inhibition of expression of SCD-1 and FAS and enhancement of ACC phosphorylation and CPT-1a expression. (g,h) Densitometric quantification of Western blot data shown in (f). Data are presented as mean +/- SEM ( n = 6). * p <0.05, ** p <0.01, *** p <0.001 vs db/db; # p <0.05, ## p <0.01 vs FGF21 WT treatment, unpaired t -test.

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Injection, Control, Staining, Western Blot, Inhibition, Expressing, Phospho-proteomics

Journal: EBioMedicine

Article Title: Paracrine-endocrine FGF chimeras as potent therapeutics for metabolic diseases

doi: 10.1016/j.ebiom.2019.09.052

Figure Lengend Snippet: . Effect of the FGF1 ΔHBS -FGF21 C-tail Chimera on Metabolic Regulation in Cynomolgus Monkeys with T2D (a) Study Design. A total of 6 spontaneous diabetic cynomolgus monkeys (4 male and 2 female) were included in this study. The first dose of FGF1 ΔHBS -FGF21 C-tail chimera was administered on day 0. (b) Changes from baseline values following administration of FGF1 ΔHBS -FGF21 C-tail for fasting blood glucose. Arrows in (b) indicate the times at which glucose tolerance tests were done. (c,d) Changes in glucose levels (c) and plasma insulin (d) during OGTTs done following administration of a low or high dose of chimera. (e) Percent change in HbAIc between day 0 and following 15 days of treatment with a low or high dose of chimera. (f,g) Changes of plasma TCHO (f) and LDL (g) lipoprotein profiles in animals treated with chimera FGF1 ΔHBS -FGF21 C-tail between before treatment and after administration of a low or high different doses. Data are presented as mean +/- SEM ( n = 3). Significance is indicated by P values comparing levels before and following administration of different doses of chimera (unpaired t -test).

Article Snippet: Blood aliquots (200 μL) were drawn from the tail vein at various times over the course of 9 h, and specific protein levels were measured using human FGF21 and FGF1 immunoassay ELISA kits (R&D Systems, Minneapolis, MN).

Techniques: Clinical Proteomics

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: Verifying the potential binding of HSPA8 with OC. A Coomassie blue staining of HSPA8. (A) total of 3 µg of purified HSPA8 was loaded for SDS-PAGE followed by Coomassie blue staining. (B) Biacore SPR analysis of HSPA8 and OC binding. HSPA8 was immobilized on the CM5 chip and different concentrations of OC passed through the chip for 60 s. The curves indicate the potential binding between OC and HSPA8. (C) The KD value was calculated from Biacore SPR data. (D) ITC assay of HSPA8 and OC was performed at 25°C. The raw ITC data for injecting OC into the sample cell containing HSPA8 protein. The reaction heat of OC and protein binding was expressed as differential power (DP) between the reference and sample cells. (E) Experimental data are expressed with solid dots and fitted to a binding curve by a one-site binding model. (F) Thermal shift assay of HSPA8 and OC. HSPA8 was incubated with different concentrations of OC, and the melting curve was plotted and calculated using fluorescence intensity.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Binding Assay, Staining, Purification, SDS Page, Isothermal Titration Calorimetry, Protein Binding, Thermal Shift Assay, Incubation, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: Verifying the potential binding of HSPA8 with OC_biotin. (A) Biacore SPR analysis of HSPA8 and OC_biotin potential binding. HSPA8 was immobilized on the CM5 chip and different concentrations of OC_biotin were passed through the chip for 60 s. The curves indicated the potential binding between OC_biotin and HSPA8. (B) The KD value was calculated from Biacore SPR data. (C) ITC measurement of HSPA8 and OC_biotin was carried out at 25°C. The raw ITC data for injecting OC_biotin into the sample cell containing HSPA8 protein. The reaction heat of OC_biotin and protein binding were expressed as differential power (DP) between the reference and sample cells. (D) Experimental data are expressed with solid dots and fitted to a binding curve by a one-site binding model.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Binding Assay, Protein Binding

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: Verifying the potential binding between OC_biotin and HSPA8 in cells. (A) OC_biotin or OC induced GFP-LC3 puncta in HeLa cells. HeLa cells with stable expression of GFP-LC3 were treated with 10 µM OC or 10 µM OC_biotin for 6 h, and GFP fluorescence was detected and analyzed using a high content screening system. Scale bar, 50 µm. (B) Statistical analysis of the number of GFP puncta formed per cell. Data are expressed as mean ± standard deviation of three independent experiments compared with control (** p < 0.01, *** p < 0.001 vs. control). (C) Western blot of LC3B, p62, and PARP. HeLa cells were treated with 10 µM OC or 10 µM OC_biotin for 24 h. The cells were lyzed using RIPA buffer and the protein expression level was analyzed by western blot. (D) Statistical analysis on the relative intensity of LC3BII, P62, cleaved PARP vs. GAPDH. GAPDH protein was used as loading control. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). (E) Immunoprecipitation of HSPA8 and OC_biotin. OC_biotin was incubated with avidin beads followed by immunoprecipitation and western blot. Upper lane: pulldown proteins eluted from avidin beads; middle band: proteins from supernatant after immunoprecipitation; lower band: input HSPA8 protein as control.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Binding Assay, Expressing, Fluorescence, High Content Screening, Standard Deviation, Control, Western Blot, Immunoprecipitation, Incubation, Avidin-Biotin Assay

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: OC and OC_biotin inhibit HSPA8 translocation in cells. (A) Colocalization of OC_biotin with HSPA8. A549 cells were treated with 10 µM OC_biotin or DMSO for 1 h. Cells were fixed with 4% paraformaldehyde and stained with streptavidin and HSPA8 antibodies. Cell nuclei were stained by DAPI. Fluorescent images were acquired using a high content screening system. (B) OC or OC_biotin blocked HSPA8 translocation following stimulation at 42°C. A549 cells were pretreated with 20 µM OC, 20 µM OC_biotin, or 20 µM PES for 1 h, then incubated at 37°C or 42°C for 4 h. The cells were then fixed and stained with HSPA8 antibody and DAPI. (C) Statistical analysis on translocation of HSPA8 from the cytosol to the nucleus. A549 cells were treated with different concentrations of OC, OC_biotin, or PES. The cells were fixed and stained with the HSPA8 antibody. The intensity ratio of the nucleus and cytosol was automatically calculated by a high content screening system. Data are expressed as mean ± standard deviation ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle control). Scale bar, 50 µm.

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Translocation Assay, Staining, High Content Screening, Incubation, Standard Deviation, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: OC promotes apoptosis upon cisplatin treatment. (A) OC increased p53 expression level when co-treated with cisplatin. A549 cells were treated with the combination of 10 µM OC, 40 µM cisplatin, and 10 µM PES for 24 h. Western blot analysis showed the changes in p53 and LC3B cleavage. (B) Statistical analysis on the relative intensity of LC3BII and P53 vs. GAPDH. GAPDH protein was used as loading control. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05 vs. OC group). (C) OC enhanced the binding between HSPA8, HSP90, and p53. A549 cells were treated with the combination of 10 µM OC, 40 µM cisplatin, or 10 µM PES for 24 h. Cell lysates were immunoprecipitated with HSPA8, and western blot analysis was performed for HSPA8, HSP90, and p53. (D) Statistical analysis on the relative intensity of HSP90 and P53 vs. HSPA8. HSPA8 protein was used as loading control. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. OC group). (E,F) OC potentiated cell death upon cisplatin treatment. A549 cells were treated with the combination of 10 µM OC, 40 µM cisplatin, or 10 µM PES for 24 h. The cells were collected and stained with Annexin V and PI for subsequent flow cytometry analysis. F shows the results of statistical analysis of apoptotic cells. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. OC group).

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Expressing, Western Blot, Control, Standard Deviation, Binding Assay, Immunoprecipitation, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro

doi: 10.3389/fphar.2020.564833

Figure Lengend Snippet: OC induces apoptosis of A549 cells by regulating HSPA8 protein. (A) Detection of the HSPA8 protein expression level in A549 cells by western blot analysis after 48 h of transfection, GAPDH was used as loading control. A549 cells were transfected with small interfering RNA (siRNA) and pCDNA_HSPA8 plasmids respectively for 48 h siRNA control (NC) and pCDNA3.1 vector (Vehicle) as a control. (B) The effects of OC on A549 cell viability after treatment with HSPA8 siRNA or pCDNA_HSPA8 plasmid. The cells were transfected with siRNA or plasmid for 48 h and OC was supplemented at the indicated concentrations for 72 h. Cell viability was then detected using the CCK-8 Assay. Data are expressed as mean ± standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control). (C) OC induces apoptosis of A549 cells by targeting HSPA8 protein. A549 cells were transfected with HSPA8 siRNA or plasmid for 48 h, 10 µM OC was supplemented for another 24 h. The cells were collected and stained with Annexin V and PI for flow cytometry analysis. (D) The result of statistical analysis of apoptotic cells. Data are expressed as mean ± standard deviation of three independent experiments (** p < 0.01, *** p < 0.001 vs. OC control group).

Article Snippet: Full-length HSPA8 (pcDNA5/FRT/TO HIS HSPA8, Addgene, Cat # 19,541) or cathepsin B (hCathepsin B, Addgene, Cat # 11,249) was subcloned into the pET-15b vector via Nco I- BamH I sites. his_HSPA8 and his_hCathepsin B were expressed in Escherichia coli BL21 (DE3) cells, respectively.

Techniques: Expressing, Western Blot, Transfection, Control, Small Interfering RNA, Plasmid Preparation, CCK-8 Assay, Standard Deviation, Staining, Flow Cytometry

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: Proteomics analysis of ROR1, ROR2, and PTK7 transfected BaF3 cells. a Principal component analysis (PCA) showing phenotypic similarity between BaF3-ROR1 and BaF3-ROR2 biological replicates, but not among the parental BaF3 and BaF3-PTK7 clones. b Heatmap of differentially expressed proteins (DEP) log 2 fold change with respect to the parental BaF3 samples. The DEPs shared among BaF3-ROR1, -ROR2, and -PTK7 are shown. c Volcano plots reporting the log 2 fold change of the DEPs between the parental BaF3 cells and BaF3 clones. Adjusted p value < 0.05 and log 2 FC >|0.5|. d Similarity matrix (shown as a heatmap) among GO biological processes related to DEPs of each BaF3 clone. The apoptotic- and metabolic-related processes are highlighted. The heatmaps in b and d are clustered using complete-linkage hierarchical clustering based on Euclidean distances

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Transfection, Clone Assay

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: Functional analysis of BaF3-ROR1, BaF3-ROR2, and BaF3-PTK7 clones. a Migration assay of BaF3 clones toward CXCL12 chemokine for 6 h. The graph shows the relative number of migrated cells normalized to BaF3 parental migrated cells. Data are represented as mean ± standard deviation (SD) and statistical comparison was performed with Student’s t test ( n = 3). b Relative survival of BaF3 cells cultured in starvation media (no serum and no IL-3) in the presence or absence of Wnt5a (100 ng/ml) for 24 h. The graph shows mean ± SD ( n = 3). In a and b, the statistical significance is indicated as * p < 0.05 and *** p < 0.001. c Immunoblotting analysis of the intracellular signaling in BaF3 parental cells and BaF3 clones expressing ROR1, ROR2, or PTK7. β-tubulin was used as a loading control. d Immunoblotting analysis of the cytoplasmic and nuclear lysates of BaF3-ROR1i clones that acquired IL-3 independence. Antibodies against the effectors of the pro-survival signaling were used to detect the protein levels and β-tubulin was used as a loading control for the cytoplasmic lysates, whereas PARP was used as a loading control for the nuclear lysates. e The relative proliferation of BaF3 parental and BaF3-ROR1i clone stimulated with 100 ng/ml Wnt5a or Wnt16b. Proliferation was measured at the indicated time points and the values were normalized to non-stimulated cells (set as value 1). The graph shows the mean ± SD ( n = 3). Statistical comparison between the non-stimulated and Wnt-stimulated BaF3-ROR1i cells was done using Student’s t test as * p < 0.05

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Functional Assay, Clone Assay, Migration, Standard Deviation, Comparison, Cell Culture, Western Blot, Expressing, Control

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: Characterization of the C-terminal cytoplasmic region of ROR1 and ROR2. a Schematic representation of amino acid boundaries of ROR1 and ROR2 C-terminal deletions (Δ1–Δ4) and the full-length (FL) receptors stably transfected into BaF3 cells. b Immunoblot analysis of the HA-tagged ROR1 and ROR2 FL and Δ1–Δ4 deletions expressed in BaF3 cells cultured for 24 h in starvation media. β-tubulin was used as a loading control and the signal quantifications shows the normalized values using BaF3 (set as 1). c Immunoblotting analysis of downstream signaling of BaF3-ROR1 and BaF3-ROR2 (FL and Δ1–Δ4) clones. β-tubulin was used as a loading control and signal quantifications show the normalized values using BaF3 samples. d Left: schematic representation of the cytoplasmic ROR1 constructs used for recombinant protein production. Expression levels of the His-tagged constructs were verified by immunoblotting. Right: DSF analysis of recombinant ROR1 constructs. Melting temperatures (T m ) are also indicated, and the data represent the mean ± SD ( n = 3). JAK2 PK domain and MuSK kinase domain was used as a reference control. e Cellular thermal shift assay (CETSA) showing stabilization of ROR1 (FL and Δ1–Δ4) upon treatment with GZD824 (10 µM). Cell lysates were heated to the indicated temperatures and immunoblotted with anti-HA for ROR1 detection. β-tubulin was used as a loading control. The graph shows the signal quantification of 3 independent experiments (mean ± SD). Band intensities were normalized to the 42 °C untreated samples for each ROR1 construct (set as 1)

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Stable Transfection, Transfection, Western Blot, Cell Culture, Control, Clone Assay, Construct, Recombinant, Expressing, Thermal Shift Assay

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: ROR1 interactome detected by BioID and AP-MS. a Venn diagram showing the number of specific ROR1 interactors identified with BioID (red), AP-MS (pink), or both (white). The respective physical interactomes and enrichment p values were obtained with STRING (reference as reported in the methods chapter). b List of selected GO biological processes and molecular functions associated with the ROR1 interactors and identified with both BioID and AP-MS

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Protein-Protein interactions

Journal: Cellular and Molecular Life Sciences

Article Title: New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

doi: 10.1007/s00018-022-04301-6

Figure Lengend Snippet: GZD824 inhibits downstream signaling of ROR1. a Schematic representation of GZD824 binding to ROR1 PK domain, which induces inhibition of its downstream PI3K/AKT, STAT3, and NF-κB signaling. b Immunoblot analysis of the BaF3-ROR1 cells left untreated of pre-treated with Src inhibitor dasatinib or GZD824 (1 µM) for 2 h before the addition of Wnt5a (100 ng/ml) as indicated. Downstream ROR1 signaling levels or pERK/ERK, pAKT/AKT, and pSrc/Src are shown. β-tubulin was used as a loading control. c Venn diagram showing the number of shared and specific interactors of ROR1 before and after GZD284 treatment as identified by AP-MS, BioID, or both. For each set of interactors, the protein-coding genes involved in MAPK signaling (red), Rho GTPases signaling (yellow), or actin/cytoskeleton signaling (blue) are indicated. d , e Bar graphs of the top non-redundant enriched ontology clusters from multiple functional annotation databases in ROR1 interactomes identified with both AP-MS and BioID when before ( d ) and after ( e ) GZD824 treatment. The color scale represents statistical significance

Article Snippet: For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), pERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA).

Techniques: Binding Assay, Inhibition, Western Blot, Control, Protein-Protein interactions, Functional Assay