Journal: PLoS ONE

Article Title: HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

doi: 10.1371/journal.pone.0077972

Figure Lengend Snippet: ( A ) GST and GST-Tat were purified on glutathione-Sepharose beads. The beads were boiled to elute the bound proteins, which were then run on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (left panel). GST pulldown assay with SK-N-MC neuroblastoma cell lysates shows a strong interaction between APP and GST-Tat (right panel). SK-N-MC neuroblastoma cell extracts incubated with GST- or GST-Tat-coated beads for 3 hours. The beads were washed three times with PBS and the eluted proteins were analyzed by western blotting with an anti-APP antibody (22C11). ( B ) Coimmunoprecipitation of Tat and APP in HEK 293FT cells transfected with Tat and/or Myc-tagged APP695 vectors. Proteins were precipitated with anti-Tat or anti-APP (6E10) antibodies and immunoblotted with anti-Tat or anti-Myc antibodies. ( C ) Coimmunoprecipitation of U-87 MG cell lysates transduced with mock, Lenti-Tat, or Lenti-mTat virus. APP was precipitated with an APP antibody (6E10), and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (22C11) or anti-Tat antibodies. Reciprocally, Tat was precipitated with anti-Tat antibody, and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (A8717) or anti-Tat antibody. ( D ) Purified recombinant APP interacts with GST-Tat. Purified recombinant APP (500 ng) was incubated with GST- or GST-Tat-coated beads and the eluted proteins were analyzed by western blotting with an APP antibody. A large amount of recombinant APP bound to the GST-Tat beads. ( E ) Tat interacts strongly with APP. SK-N-MC neurobalstoma cell lysates were incubated with GST, GST-Tat, or GST-Tat beads, and washed three times in buffer containing 137, 200, 300, 400 or 500 mM NaCl. APP remained associated with GST-Tat under high-salt conditions. ( F ) The cysteine-rich domain of Tat is important for association with APP. Deletion mutants were produced as GST-fusion proteins and subjected to GST-pulldown assays with SK-N-MC cell lysates. L, load; B; bound.

Article Snippet: Purified recombinant APP (cat. no APP-526H, Creative BioMart, NY, USA) was resuspended in PBS supplemented with 1% NP-40 to yield a final concentration of 1 ng/μl, and 500 μl was incubated with GST- or GST-Tat-coated beads for 3 hours at 4°C.

Techniques: Purification, SDS Page, Staining, GST Pulldown Assay, Incubation, Western Blot, Transfection, Transduction, Virus, Recombinant, Produced

Journal: Nature Communications

Article Title: Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration

doi: 10.1038/s41467-022-32944-3

Figure Lengend Snippet: a Western blot analysis of p38α, phosphorylated-p38 (p-p38), and downstream substrates MK2 and MNK1 in tissue homogenates of the brain cortex after 2 weeks of either vehicle or NFMD treatment ( n = 5 for Ts2-NFMD, and n = 6 for other treatment groups). b Quantification of the western blot images shown with Image J and graphed with GraphPad Prism 8.0.1 [Ordinary One-Way ANOVA, For p-p38 F(3, 19) = 6.658, R square = 0.540, for 2 N vs Ts2, p = 0.0044, 95% CI = −0.760, −0.131; for 2 N vs Ts2-NFMD p = 0.0495, 95% CI = −0.656 −0.000194; For MK2 F(3, 19) = 10.33, R square = 0.610, for 2 N vs Ts2-NFMD, p = 0.0005, 95% CI = 0.182, 0.541; for Ts2 vs Ts2-NFMD p = 0.0166, 95% CI = 0.0457, 0.404; For pMK2 F(3, 19) = 2.850, R square = 0.310, for 2 N vs Ts2, p = 0.0178, 95% CI = −1.095, −0.117; for 2 N vs 2N-NFMD p = 0.0469, 95% CI = −0.985, −0.0076; for Ts2 vs Ts2-NFMD p = 0.0166, 95% CI = 0.0457, 0.404; For MNK1 F(3, 19) = 15.11, R square = 0.705, for 2 N vs Ts2, p = 0.0003, 95% CI = 0.235, 0.668; for 2 N vs 2N-NFMD p = 0.023, 95% CI = 0.0393, 0.472, for Ts2 vs Ts2-NFMD, p = 0.0347, 95% CI = 0.0197, 0.473; For pMNK1 F(3, 19) = 6.285, R square = 0.498, for 2 N vs Ts2-k NFMD, p = 0.0063, 95% CI = 0.156, 0.824; for TS2 vs Ts2-NFMD p = 0.0428, 95% CI = 0.125, 0.681]. c , d Subsequent western blot analysis for full length APP (APPfl), APP-βCTF and BACE1 and western blot image quantified with Image J and graphed with GraphPad Prism 8.0.1 (Ordinary One-Way ANOVA, For APPfl F(3, 19) = 23.33, R square = 0.786, for 2 N vs Ts2, p < 0.0001, 95% CI = −684, −0.376; for 2 N vs Ts2-NFMD p = 0.0001, 95% CI = −0.532, −0.209; For APP-βCTF F(3, 19) = 8.167, R square = 0.563, for 2 N vs Ts2, p = 0.0003, 95% CI = −1.027, −0.367; for Ts2 vs Ts2-NFMD p = 0.0007, 95% CI = 0.3187, 1.010; For BACE1 F(3, 19) = 12.86, R square = 0.670, for 2 N vs Ts2-NFMD, p = 0.0001, 95% CI = 0.158, 0.491; for Ts2 vs TS2-NFMD p = 0.0469, 95% CI = −0.00185, 0.335). Data are presented as mean values ± SEM. Statistical significance is represented by asterisks * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Source data are provided as a Source Data File.

Article Snippet: For protein analyses, mouse brain tissues were homogenized and western blot analyses were performed with antibodies against APP (c1/6.1; 1:1000), bCTF (M3.2, 1:250) , BACE1 (Rockland; 200-401-984; 1:500), MAPKAPK-2 (MK2; Cell Signaling; 12155; 1:500), phospho-MK2 (Cell Signaling; 3007; 1:500), p38 MAPK (p38α; Cell Signaling; 9218; 1:500), phosphor-p38 (Santa Cruz; 166182; 1:500), MNK1 (Cell Signaling; 2195; 1:500), pMNK1 (Cell Signaling; 2111; 1:500), β-actin (Santa Cruz Biotechnology; sc-47778; 1:2000).

Techniques: Western Blot

Journal: Biomarker Research

Article Title: Single-cell and spatial transcriptome analysis reveals the potential therapeutic targets for testicular sex cord-stromal cell tumor

doi: 10.1186/s40364-026-00924-0

Figure Lengend Snippet: Disordered cellular communication in TSCST. ( A ) Bar plot showing the number of inferred interactions in the leading edge and the tumor core. ( B ) Bar plot showing the interaction strength in the leading edge and the tumor core. ( C ) Circle plots display ligand-receptor interaction weights between distinct cellular components in the leading edge and the tumor core. Colors distinguish cell types. The circle size is proportional to the number of cells in each cluster, and the edge width represents communication probability. ( D ) Dot plots show the increased ligand-receptor interactions on the leading edge. ( E ) Dot plots showing the increased ligand-receptor interactions in the tumor core. ( F ) Dot plots show increased signaling in macrophages to tumor and Sertoli cells. ( G ) Feature plots of APP-CD74 by ST in Patient 3 and Patient 4. ( H ) Feature plots of MDK-NCL by ST in Patient 3 and Patient 4. ( I ) Mapped SPP and MK signaling pathways in ST, colored by Seurat clusters. ( J ) Violin plots showing the expression level of APP-CD74 genes across the cell types in the leading edge and the tumor core, respectively. The y-axis shows the normalized read count. ( K ) Representative immunofluorescence images showing expression of CTNNB1 and CD74 (n = 4). Nuclei were stained with DAPI. ( L ) Relative mRNA expression of MRC1 and ARG1 (M2-type macrophage markers) and IL1B (M1-type macrophage marker) in macrophages treated with APP. ( M ) Relative mRNA expression of MMP2 (a marker associated with phagocytic function in macrophages) in macrophages treated with APP. The P value was calculated by Welch’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: THP-1-induced macrophages treated with APP protein (MedChemExpress, Shanghai, Catalogue number: HY- P72834 ) were collected, and total RNA was extracted using TRIzol Reagent (Invitrogen, 15596018CN, USA).

Techniques: Protein-Protein interactions, Expressing, Immunofluorescence, Staining, Marker