db987 against full length recombinant human pde9  (Sino Biological)

Journal: Pharmaceutical Biology

Article Title: Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

doi: 10.1080/13880209.2026.2646350

Figure Lengend Snippet: GAC binds HSPA8 to induce ferroptosis in HCC. (A) Schematic diagram of the drug Affinity responsive Target stability (DARTS) assay workflow. (B) Representative coomassie blue-stained SDS-PAGE gel of DARTS samples. (C) Bubble plot summarizing MS results: x-axis shows δsequence coverage (GAC vs. control), bubble size represents protein score. (D) Protein-protein interaction (PPI) network of DARTS-identified targets; nodes are colored by DMNC algorithm score (red = high centrality, yellow = low). (E–F) Cellular thermal shift assay (CETSA) confirms GAC binding stabilizes HSPA8. Representative immunoblots (E) of HSPA8 levels in lysates from HCCLM3 cells treated with GAC or DMSO and subjected to a temperature gradient (37-65 °C) and quantification (F) of relative HSPA8 levels at each temperature, data were assessed by the repeated-measures ANOVA, n = 3 in each group. (G–H) Microscale thermophoresis (MST): representative MST traces (G); dose-response curve showing GAC binding to recombinant HSPA8 (H). (I–J) molecular docking pose(I) of GAC bound to HSPA8 and magnified view of the molecular docking pose (J) focusing on the interactions between GAC and key residues (Tyr15, Gly201, Arg272, Asp366). (K–M) molecular dynamics (MD) simulations of GAC bound to HSPA8: RMSD of HSPA8 backbone over 100 ns (K); RMSF per residue(L); per-residue energy decomposition identifying key interacting residues (M). (N) Schematic of HSPA8 point mutations generated at predicted GAC-binding residues. (O-P) CETSA validation of HSPA8 mutants: Western blots (O) and quantification (P) demonstrate loss of thermal stabilization by GAC in HSPA8 MUT , n = 3 in each group. (Q–R) Flow cytometry histogram of Bodipy665/676 intensity and quantification of the percentage of oxidized cells in shHSPA8 vs. scramble cells treated with GAC or not, n = 3 in each group. (S–T) Knock down HSPA8 restores redox homeostasis: Quantified MDA levels, data were assessed by the Mann-Whitney test. (S) and GSH/GSSG ratio (T), n = 3 in each group. (U-V) Flow cytometry plots (U) and quantification (V) of the percentage of Annexin V + cells in shHSPA8 vs. scramble cells treated with GAC or not, n = 3 in each group. (W-X) Knock down HSPA8 ameliorates clonogenic capacity: Representative colony formation images(W) and quantified colony counts (X), n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified HSPA8 (11329-H07E, sinoBiological) served as a target and was labeled with fluorescence by Monolith Labeling Kit RED-tris-NTA (MO-L018, NanoTemper Technologies).

Techniques: Staining, SDS Page, Control, Thermal Shift Assay, Binding Assay, Western Blot, Microscale Thermophoresis, Recombinant, Residue, Generated, Biomarker Discovery, Flow Cytometry, Knockdown, MANN-WHITNEY

Journal: Pharmaceutical Biology

Article Title: Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

doi: 10.1080/13880209.2026.2646350

Figure Lengend Snippet: GAC promotes CMA-dependent GPX4 degradation to induce ferroptosis. (A–B) GAC induces HSPA8-dependent KFERQ puncta formation, indicating CMA activation. Representative images of confocal images (A) detecting the KFERQ-probe (bar = 25 μm) and quantification of KFERQ puncta per cell (B) using ImageJ, n = 3 in each group. (C) CCK-8 assay assessing cytotoxicity in cells pretreated with indicated concentrations of Bafilomycin A1 (BafA1) for 12 h before GAC treatment, n = 3 in each group. (D–E) Flow cytometry histogram of Bodipy-C11 intensity (D) and quantification (E) of the percentage of oxidized cells in HCCLM3 cells pretreated with BafA1 before GAC treatment, n = 3 in each group. (F–G) Flow cytometry plots (F) and quantification (G) of the percentage of Annexin V + cells in HCCLM3 cells pretreated with BafA1 before GAC treatment, n = 3 in each group. (H) Table generated by KFERQ-finder showing the gene names, KFERQ-like motifs, and their starting positions within GPX4 and SLC7A11. (I-K) HSPA8 knockdown specifically prevents GAC-induced GPX4 degradation. Representative immunoblots (I) and quantification of GPX4 (J) and SLC7A11(K), normalized to β-actin, n = 3 in each group. (L–N) LAMP2A knockdown abolishes GAC-induced GPX4 degradation. Representative immunoblots (L) and quantification of LAMP2A (M) and GPX4 (N), normalized to β-actin, n = 3 in each group. (O–P) Flow cytometry histogram of Bodipy-C11 intensity (O) and quantification (P) of the percentage of oxidized cells in shLAMP2A vs. scramble cells treated with GAC or not, n = 3 in each group. (Q-R) Flow cytometry plots (Q) and quantification (R) of the percentage of Annexin V + cells in shLAMP2A vs. scramble cells treated with GAC or not, n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified HSPA8 (11329-H07E, sinoBiological) served as a target and was labeled with fluorescence by Monolith Labeling Kit RED-tris-NTA (MO-L018, NanoTemper Technologies).

Techniques: Activation Assay, CCK-8 Assay, Flow Cytometry, Generated, Knockdown, Western Blot

Journal: Pharmaceutical Biology

Article Title: Ginkgolic acid targets HSPA8 to trigger ferroptosis in hepatocellular carcinoma via chaperone-mediated autophagy-dependent GPX4 degradation

doi: 10.1080/13880209.2026.2646350

Figure Lengend Snippet: GAC binds HSPA8 to promote HSPA8-GPX4 interaction via KFERQ-like motif. (A) GAC enhances the endogenous HSPA8-GPX4 interaction. Co-immunoprecipitation (co-IP) of GPX4 followed by immunoblotting for HSPA8 in HCCLM3 (left panel) and HepG2 (right panel) cells, n = 3 in each group. (B-D) GAC increases HSPA8-GPX4 co-localization. Representative immunofluorescence confocal microscopy images (B) showing endogenous GPX4 (red) and HSPA8 (green). Nuclei were stained with DAPI (blue). Scale bar: 25 μm. Line-scan intensity profiles (C) across representative regions of interest (ROIs) from (B), demonstrating correlated fluorescence signals of HSPA8 and GPX4 at subcellular resolution. Quantification (D) of co-localization using Pearson’s correlation coefficient (analyzed with the JACoP plugin in ImageJ), n = 3 in each group. (E) Co-IP of GPX4 in cells expressing wild-type HSPA8 (HSPA8 WT ) or binding-deficient mutant HSPA8 (HSPA8 MUT ); GAC enhances HSPA8-GPX4 interaction only in the presence of HSPA8 WT .(F) schematic of GPX4 highlighting two critical KFERQ-like motifs (AAKFD and LIDAA) targeted for mutagenesis to generate GPX4 AAKFD and GPX4 LIDAA . (G, H) Immunoblotting (G) and quantification (H) showing that GAC induces degradation of wild-type GPX4 but not KFERQ-mutant GPX4 in HCCLM3 cells. (I) Co-IP assay demonstrating loss of interaction between HSPA8 and KFERQ-mutant GPX4 upon GAC treatment. (J, K) Overexpression of HSPA8 promotes degradation of Myc-GPX4 WT but not Myc-GPX4 AAKFD : representative immunoblots (J) and quantification (K) of GPX4, normalized to β-actin, n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified HSPA8 (11329-H07E, sinoBiological) served as a target and was labeled with fluorescence by Monolith Labeling Kit RED-tris-NTA (MO-L018, NanoTemper Technologies).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence, Expressing, Binding Assay, Mutagenesis, Over Expression

Journal: Biomaterials Science

Article Title: Modular noncovalent functionalization of electrospun piezoelectric scaffolds with bioactive nanocarriers

doi: 10.1039/d5bm01563d

Figure Lengend Snippet: Liposome coated scaffolds are suitable substrates for cells and enable cytokine presentation: (A) live HEK293 cell counts were measured from multiple time points on unpoled scaffolds with and without cationic liposome coating. Data is presented as mean ± SEM from 3 independent wells at each time point. Statistical significance was assessed by two-way ANOVA with Sidak's multiple comparisons test, comparing coated to uncoated scaffold at each time point. P values reflect differences between groups at individual time points, and all error bars are present on graph although some are too small to see. (B) Unpoled scaffolds were coated with fluorescent Cy5-labeled liposomes and imaged with confocal microscopy. Jurkat T cells were stained with calcein, cultured on Cy5 liposome-coated scaffolds, and imaged at 60× resolution 3 days after plating. (C) Jurkat T cells were fixed on day 4 and stained intracellularly with ActinProbe555 and DAPI. Arrows indicate punctate actin (yellow) or liposome uptake (magenta). Scale bars are 50 µm or 10 µm. (D) Activating the JAK-STAT Pathway with IL-15 Liposomes on Scaffolds: (i) unpoled scaffolds were coated with 2 mg mL −1 of IL-15-tethered liposomes in optimized HEPES-NaCl buffer conditions and SEAP is produced in response to IL-15 recognition. (ii) IL-15 reporter cells were plated on unpoled scaffolds, and absorbance was read daily, correlating to the downstream SEAP production from activation of the JAK-STAT pathway with n = 3 samples per group. Data is presented as relative fold change from uncoated scaffold control, with mean ± SD. Significance is conducted with an Ordinary Two-Way Anova with multiple comparisons using Šídák's multiple comparisons test.

Article Snippet: Liposomes were incubated with recombinant histidine-tagged human IL-15 (Sino Biological #10360-H07E) overnight at 4 °C to achieve a concentration of 180 ng mL −1 IL-15 on liposomes at 2 mg mL −1 , the loading concentration, in HEPES-NaCl.

Techniques: Labeling, Liposomes, Confocal Microscopy, Staining, Cell Culture, Produced, Activation Assay, Control

Journal: Nature Communications

Article Title: Nanopore-based massively parallel sensing for peptide profiling and protein identification

doi: 10.1038/s41467-026-69628-1

Figure Lengend Snippet: a Confusion matrix for the classification of SAAVs based on amino acid charge and volume. The “others” category represents the aggregation of all uncharged amino acids excluding K, R, D, and E. And S, M, and L represent small (A, C, G, N, P, S and T), medium (V, H and Q) and large (I, L, M, F, Y, and W) amino acid categories within SAAVs, respectively . b Classification for SAAVs involving leucine (L) and isoleucine (I), and their corresponding DMs for “3I” and “3L”. c Classification of positional variants involving the charged amino acid D and the bulky amino acid Y, along with their corresponding DMs. d Confusion matrix for the classification of PTMs (phosphorylation, acetylation, methylation, and octanoylation) on the serine residue of 1S peptide (GSGS S SGSGK). e Illustration of classification and corresponding DMs induced by potential natural modifications and mutations at specific sites within a model protein, hp1 and hp3.

Article Snippet: GABARAPL2, referred to as hp3, was commercially obtained from Sino Biological, Inc. (Cat: 14563-H07E).

Techniques: Phospho-proteomics, Methylation, Residue

Journal: Nature Communications

Article Title: Nanopore-based massively parallel sensing for peptide profiling and protein identification

doi: 10.1038/s41467-026-69628-1

Figure Lengend Snippet: a The confusion matrix of the test set based on the CNN-DM filtered dataset. Label hp1_1 represents the first LysC-derived peptide fragment from the hp1 protein (etc.). Data with an error rate lower than 1% is not shown. b Schematic of the single-blind protein identification workflow. Anonymously labeled protein samples (Protein 1, 2, or 3) are individually subjected to LysC digestion. The resulting peptide mixtures are then azidated with FSO 2 N 3 , followed by OPO library preparation. Nanopore sensing and CNN-DM analysis then allow for the assessment of their distribution characteristics and the prediction of the protein’s identity as one of three candidates (hp1, hp2, or hp3). c Distribution of predicted OPO reads for three model proteins generated from CNN-DM-based classification of protein 1 (dark green), protein 2 (yellow), and protein 3 (light green), which were identified as hp1 ( n = 9 peptides), hp2 ( n = 6 peptides) and hp3 ( n = 9 peptides), respectively. The Tukey box plot summarizes OPO reads’ distribution, where the box represents the interquartile range (IQR) from the 25th to the 75th percentile, the central line indicates the median, the whiskers extend to the highest and lowest values within 1.5 times the IQR, and any data points beyond the whiskers are identified as outliers.

Article Snippet: GABARAPL2, referred to as hp3, was commercially obtained from Sino Biological, Inc. (Cat: 14563-H07E).

Techniques: Derivative Assay, Labeling, Generated

Journal: Nature Communications

Article Title: Loss of Fsr quorum sensing promotes biofilm formation and worsens outcomes in enterococcal infective endocarditis

doi: 10.1038/s41467-026-68366-8

Figure Lengend Snippet: A Z-projections of WT and Δ gelE vegetations at 72 hpi, captured with LSCM and stained for DNA, myeloperoxidase (MPO), and IL-1β. Cyan insets are highlighting the presence of IL-1β between the NETs-biofilm interface. Orange inset is shown in Fig. S4B, highlighting the colocalization of IL-1β with neutrophils. Representative images shown from n = 3, N = 1. Bf = biofilm, scale = 20 µm. B Detection and quantification of IL-1β in WT and Δ gelE vegetations at 72 hpi with western blotting. Loading control = β-actin. Mean ± SEM, n = 5 from N = 2, A.U. = arbitrary units. Statistical significance was assessed with a two-tailed t-test. C Rat pro-IL-1β and its cleaved fragments in supernatants from OG1RF WT and mutant strains detected by western blotting. Rat pro-IL-1β was incubated in BHI with indicated OG1RF strains for 2, 4, 6 h. BHI = Negative control with only media and pro-IL 1β. Representative blot shown from N = 2. D Human pro-IL-1β and its cleaved fragments in supernatants from OG1RF WT and mutant strains detected by western blotting. Human pro-IL-1β was incubated in BHI with indicated OG1RF strains for 2, 4, 6, and 24 h. Gelatinase presence was also determined in these supernatants. Δ gelE :: gelE E352A expresses proteolytically inactive gelatinase. Representative blot shown from N = 2. BHI = Negative control with only media and pro-IL 1β. E Mean activation of HEK-Blue IL-1R reporter cells by supernatants harvested from OG1RF WT and Δ gelE :: gelE E352A cultures with or without human pro-IL-1β at 18 h. Stimulation of cells with mature human IL-1β was used as a positive control. N = 3, Error = SEM. Statistical significance was determined with one-way ANOVA. F Schematic representation of gelatinase (blue) and caspase-1 cleavage sites (red) across human, rat, and mouse pro-IL-1β; n = animals per group, N = independent experiments, ns not significant (p ≥ 0.05), Red arrowhead = pro-IL-1β, blue arrowhead = mature IL-1β. Exact p values are reported in the figure. Source data are provided as a Source Data file.

Article Snippet: E. faecalis overnight cultures of OG1RF WT and mutants were diluted 1:10 in 200 μl fresh BHI supplemented with human pro-IL-1β (100 ng/mL; Cat. Nr. 10139-H07E, Sino Biological) and incubated for 2, 4, 6 and 24 h, or with rat pro-IL-1β (10 ng/ml, Cat. Nr. 80023-R07E, Sino Biological) and incubated at 2, 4, and 6 h at 37 °C, static conditions.

Techniques: Staining, Western Blot, Control, Two Tailed Test, Mutagenesis, Incubation, Negative Control, Activation Assay, Positive Control