pp2a blocker lb  (MedChemExpress)

Journal: Journal of Biomedical Science

Article Title: SARS-CoV-2 membrane protein recruits PP2A to dephosphorylate the nucleocapsid and promote virion production

doi: 10.1186/s12929-026-01255-w

Figure Lengend Snippet: The Involvement of the Phosphatase PP2A in M-Mediated N Dephosphorylation and Particle Release. A , B Cell lysates from transfected 293T cells expressing the indicated viral proteins and treated with pan-PPase inhibitors (OA, okadaic acid; Tau, tautomycin) or PP2A-specific inhibitors (Fos, fostriecin; LB, LB-100) were analyzed by western blotting to evaluate the phosphorylation status of the N protein. C 293T cells co-expressing N and M proteins, with lentiviral knockdown of the catalytic subunit of PP2A (shPPP2C, left panels) or PP1 (shPPP1C, right panels), were lysed and analyzed by western blotting to assess N phosphorylation status. The pN ratios (phosphorylated N normalized to total N protein) were compared, with N expressed alone and the shLuc lentivirus-infected control set to 100%. D VLP titers in the culture media of producing 293T cells (co-transfected with N/M/E/S-expressing constructs and the packable T20-Luc RNA clone), with or without knockdown of the PP2A catalytic subunit (shPPP2C vs. shLuc control), were quantified by RT-qPCR. Data are presented as mean ± SD from three independent experiments (left bar graph). Cell lysates were analyzed in parallel by western blotting to confirm viral protein expression and knockdown efficiency (right panels). **p < 0.01. E The interaction of N and M proteins with the PP2A catalytic subunit was examined by co-immunoprecipitation using anti-N antibodies in lysates from 293T cells co-expressing N and M (48 h post-transfection). F Distribution of SARS-CoV-2 structural proteins (N, M, and S) and the PP2A catalytic subunit across fractions 5–12 was analyzed by western blotting. The yellow region indicates fractions 11–12 containing VLP-like particles, in which dephosphorylated N, PP2A, and T20-Luc RNA are detected. RNA titers and densities of the individual fractions are shown at the bottom of each lane

Article Snippet: Another pan-PPase inhibitor tautomycin and PP2A blocker LB-100 were ordered from MedChemExpress.

Techniques: De-Phosphorylation Assay, Transfection, Expressing, Western Blot, Phospho-proteomics, Knockdown, Infection, Control, Construct, Quantitative RT-PCR, Immunoprecipitation

Journal: Journal of Biomedical Science

Article Title: SARS-CoV-2 membrane protein recruits PP2A to dephosphorylate the nucleocapsid and promote virion production

doi: 10.1186/s12929-026-01255-w

Figure Lengend Snippet: The M Protein Cooperates with Phosphorylated N to Recruit PP2A to the ERGIC for N Dephosphorylation. A 293T cells co-expressing N and M proteins were lysed for cytosol/membrane fractionation, followed by western blot analysis. B Isotonic lysates prepared from N/M-expressing 293T cells were subjected to sequential centrifugation at differential speeds. The final sedimented membrane fractions and concentrated supernatants were analyzed by western blotting. C , D N/M-expressing 293T cells were homogenized in isotonic buffer, and cell lysates were fractionated on a 10–50% sucrose gradient by ultracentrifugation. Equal volumes of fractions 4–13 were analyzed by western blotting ( C ). Similarly, equal volumes of ERGIC-containing fractions (F5–F8) obtained from lysates of N- or N/M-expressing 293T cells were analyzed side by side by western blotting ( D ). E , F Cytosol/membrane-fractionated lysates obtained from N/M-expressing 293T cells, either with expression of the phospho-mutant (N-S188A/S206A) ( E ) or following treatment with LY (1 μM) ( F ), were analyzed by western blotting. G As designed in ( E ) and ( F ), ERGIC-enriched lysates (i.e., the final sedimented membrane fraction obtained by sequential centrifugation) were analyzed by western blotting. ERGIC-53, RTN3, and G3BP1 served as subcellular markers for the ERGIC, ER lumen, and stress granules, respectively. FASN, PK, and GAPDH indicated cytosolic fractions. All blotting data are representative of three independent experiments

Article Snippet: Another pan-PPase inhibitor tautomycin and PP2A blocker LB-100 were ordered from MedChemExpress.

Techniques: De-Phosphorylation Assay, Expressing, Membrane, Fractionation, Western Blot, Centrifugation, Mutagenesis

Journal: Journal of Biomedical Science

Article Title: SARS-CoV-2 membrane protein recruits PP2A to dephosphorylate the nucleocapsid and promote virion production

doi: 10.1186/s12929-026-01255-w

Figure Lengend Snippet: Blocking PP2A Activity Suppresses SARS-CoV-2 Virion Production and Decreases pgRNA Transcription. A Schematic illustration of the procedure used to examine the inhibitory effects of PP2A inhibitors on VLP release in the 293T culture system (upper diagram). VLP titers in culture supernatants collected from 293T cells treated with fostriecin (Fos) or LB-100 (LB) were determined by RT-qPCR. Data were obtained from three independent experiments and are presented as mean ± SD, with the ddH₂O-treated group set to 100% (lower bar graphs). B Similar to ( A ), a schematic of the protocol used to examine the antiviral effects of okadaic acid (OA) or PP2A inhibitors against SARS-CoV-2 infection in Calu-3 cells is shown (upper diagram). Culture supernatants collected at 24 h post-infection (MOI = 0.01) were analyzed by plaque assay to determine viral loads. Titers represent the average of three independent experiments and are shown as log₁₀ plaque-forming units (PFU)/mL, along with relative percentages compared to the ddH₂O-treated group (set to 100%) (lower bar graphs). C As described in ( B ), the corresponding culture supernatants were analyzed by western blotting to assess viral protein levels in released virions. D , E As described in ( C ), cell lysates and extracted RNAs from infected cells treated with OA or PP2A inhibitors were analyzed by western blotting and northern blotting, respectively, to assess Akt/GSK-3 signaling molecules ( D ), SARS-CoV-2 gRNA and individual sgRNAs ( E , upper panels), and viral proteins ( E , middle and lower panels). Relative gRNA levels determined by RT-qPCR (with PBGD as an internal control), relative levels of phosphorylated Akt, GSK-3, and N proteins (normalized to total protein), and fold changes in viral N, M, S, and Nsp3 proteins were compared with the ddH₂O-treated group (set to 1.0) and shown below. All blotting data are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Another pan-PPase inhibitor tautomycin and PP2A blocker LB-100 were ordered from MedChemExpress.

Techniques: Blocking Assay, Activity Assay, Quantitative RT-PCR, Infection, Plaque Assay, Western Blot, Northern Blot, Control

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: Alterations in PPP2R5C protein levels in NDEs from the plasma of AD patients (A) Representative TEM image of NDEs. Scale bars: 1 μm for low magnification and 100 nm for high magnification. (B) Nanoparticle tracking analysis of NDEs, confirming the expected size range. (C) Western blot analysis validation of NDEs and non-NDEs, using neuronal exosome markers, CD171 and synaptophysin; a microglia-derived exosome marker, Trem2; an astroglia-derived exosome marker, GLAST; and the exosome marker, Alix. (D) Label-free proteomic analysis of plasma NDEs showing differences in PPP2R5C protein levels among CN controls ( n = 4), pre-symptomatic familial AD mutation carriers (pre-FAD) ( n = 4), and familial AD (FAD) patients ( n = 5). (E) Targeted protein analysis comparing PPP2R5C protein levels in plasma NDEs across AD ( n = 20), aMCI ( n = 12), and CN groups ( n = 32). (F) Western blot analysis showed the difference in PPP2R5C levels in NDEs between AD, aMCI, and CN groups. All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗ p < 0.05 and ∗∗ p < 0.01 with Student’s t test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: Clinical Proteomics, Western Blot, Biomarker Discovery, Derivative Assay, Marker, Mutagenesis

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: Diagnostic utility of plasma PPP2R5C protein levels in AD (A) Plasma PPP2R5C protein levels in AD, aMCI, and CN groups by ELISA technology. AD, n = 74. aMCI, n = 76. CN, n = 74. (B) Evaluation of the diagnostic performance of plasma PPP2R5C protein levels in distinguishing AD from aMCI and CN. (C–F) Spearman correlation analysis between plasma PPP2R5C levels, Mini-Mental State Examination (MMSE) scores, and other AD biomarkers, including p-Tau T181, p-Tau S231, and p-Tau T217. (G) Representative western blots for PPP2R5C in brain tissues from young individuals, cognitively normal elderly individuals, and AD patients. (H) Quantitation of PPP2R5C levels, normalized to young CN ( n = 5 or 7). (I–K) Representative immunohistochemistry images of PPP2R5C (I), AT-8 (J), and p-Tau T181 (K) levels in the hippocampus of healthy controls and Braak-graded AD brain slices. Scale bars: 1 mm in (I, left), 50 μm in (I, right), and 1 mm in (J and K). (L) Quantitation of PPP2R5C, p-Tau T181, and AT8 levels (sample quantify, health ctr: 5, Braak II: 9, Braak III: 5, and Braak IV: 4). (M) Difference of plasma PPP2R5C protein levels among AD ( n = 34), PSP ( n = 28), and FTD ( n = 37) patients using ELISA. (N) ROC curve analysis assessing the discriminative power of plasma PPP2R5C protein levels for distinguishing AD from PSP and FTD. All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and n.s., no statistics with one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: Diagnostic Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitation Assay, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: PPP2R5C interacts with Tau and attenuates its expression (A) Co-immunoprecipitation analysis demonstrating the interaction between PPP2R5C and Tau protein. (B) Representative western blots immunostained for t-Tau, p-Tau T181, p-Tau S202, and p-Tau S396, in HEK293 cell lysates following PPP2R5C overexpression. (C) Quantification of t-Tau, p-Tau T181, p-Tau S202, and p-Tau S396, normalized to vector ( n = 6). (D) Representative western blots showing t-tau, p-Tau T181, p-Tau S202, and p-Tau S396 in primary neurons from Tau P301S mice following PPP2R5C overexpression. (E) Quantification of t-Tau, p-Tau T181, p-Tau S202, and p-Tau S396 levels, normalized to vector ( n = 6). (F and G) Double-labeling immunofluorescence analysis of GFP (green) and t-Tau/p-Tau T181 (red) was conducted on primary Tau P301S neurons after PPP2R5C overexpression. Scale bars, 10 μm. (H and I) Quantification of t-tau and p-Tau T181 fluorescence intensities ( n = 6). (J) Representative DiI staining shows the difference in spine density in tau P301S neurons after PPP2R5C overexpression. Scale bar: 10 μm. (K) Quantification of the density of spines ( n = 10). All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗∗ p < 0.01 and ∗∗∗ p < 0.001 with Student’s t test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: Expressing, Immunoprecipitation, Western Blot, Over Expression, Plasmid Preparation, Labeling, Immunofluorescence, Fluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: Overexpression of PPP2R5C in Tau P301S mice reduces AD-like pathogenesis and rescues cognitive function (A) Schematic representation of the experimental design. Two-month-old Tau P301S mice were injected with either AAV-hSyn-EGFP or AAV-hSyn-PPP2R5C-EGFP. Mice were sacrificed 6 months after AAV injection. (B and C) Morris water maze analysis as escape latency (s) and escape latency on day 4 (s). (D) Probe trial performance of Morris water maze test. (E) Swim speed of mice injected with AAVs encoding EGFP or PP2R5C-EGFP ( n = 8–10 mice per group). (F) Time spent in the novel arm in the Y-maze test ( n = 8–10 mice per group). (G, I, and K) Representative immunostaining images of t-Tau, p-Tau T181, and AT8 in the hippocampus of Tau P301S mice injected with AAVs encoding EGFP or PPP2R5C-EGFP. Scale bars: 200 μm for 4× images and 20 μm for magnified images. (H, J, and L) Quantification of immunoreactivity for t-Tau, p-Tau T181, and AT8 ( n = 5 mice per group). (M and N) Representative western blots showing Tau pathology in mouse brain tissue following PPP2R5C overexpression ( n = 6 mice per group). (O) Golgi staining revealed the dendritic spines in the apical dendritic layer of the CA1 region. Scale bar, 10 μm. (P) Quantification of spine density ( n = 6 mice per group). (Q) Representative electron microscopy of the synapse structures. Arrows indicate synapses. Scale bar, 1 μm. (R) Quantification of synaptic density ( n = 6 mice per group). All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and n.s., no statistics, Student’s t test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: Over Expression, Injection, Immunostaining, Western Blot, Staining, Electron Microscopy

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: Knockdown of PPP2R5C in Tau P301S mice worsens cognitive dysfunctions (A) Schematic representation of the experimental design. Two-month-old Tau P301S mice were injected with either AAV-sh-Ctrl-EGFP or AAV-sh-PPP2R5C-EGFP. Mice were sacrificed 4 months after AAV injection. (B and C) Morris water maze analysis as escape latency (s) and escape latency on day 4 (s). (D) Probe trial performance of Morris water maze test. (E) Swim speed of mice injected with AAVs encoding sh-Ctrl-EGFP or sh-PPP2R5C-EGFP ( n = 8–10 mice per group). (F) Time spent in the novel arm in the Y-maze test ( n = 8–10 mice per group). (G, I, and K) Representative immunostaining images of t-Tau, p-Tau T181, and AT8 in the hippocampus of Tau P301S mice injected with AAVs encoding sh-Ctrl-EGFP or sh-PPP2R5C-EGFP. (H, J, and L) Quantifying immunoreactivity for t-Tau, p-Tau T181, and AT8 ( n = 4 mice per group). Scale bars: 200 μm for 4× images and 20 μm for magnified images. (M and N) Representative western blots showing Tau pathology in mouse brain tissue following PPP2R5C knockdown ( n = 6). (O) Golgi staining revealed the dendritic spines in the apical dendritic layer of the CA1 region. Scale bar, 10 μm. (P) Quantification of spine density ( n = 6 mice per group). (Q) Electron microscopy of synapses (left) and high magnification of synapses (right). Scale bar: 2 μm in the left panel, 200 μm in the right panel. (R) Quantification of synaptic density ( n = 6 mice per group). All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and n.s., no statistics, Student’s t test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: Knockdown, Injection, Immunostaining, Western Blot, Staining, Electron Microscopy

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: PPP2R5C promotes Tau degradation and dephosphorylation by regulating autophagy and PP2A activity (A) Representative western blots show autophagy-lysosome inhibitors NH4Cl, Leu, and CQ prevent PPP2R5C-derived Tau degradation. (B) Quantifying relative Tau, p-Tau T181, p-Tau S202, and p-Tau S396 protein levels ( n = 6). (C) EM morphometric analysis of autophagic vacuoles in conditions of starvation combined with OA treatment and autophagic flux blockade combined with (PPP2R5C + LP) or without (PPP2R5C) treatment. Scale bar, 1 μm. (D) Representative IF images of AT8 and p62 after Tau P301S primary neurons infected with GFP-PPP2R5C/shPPP2R5C lentivirus. Scale bar, 10 μm. (E) Quantification of fluorescence density of p62 in neurons. ( n = 10). (F and H) Representative IF images of SH-SY5Y cells co-transfected with tandem mCherry-GFP-tagged autophagy marker LC3 plus empty vector or FLAG-PPP2R5C or siPPP2R5C. Autophagosomes were visualized with yellow dots (mCherry and GFP co-localization) and autolysosomes were identified as red dots (mCherry). Scale bars: 10 μm in normal and 2 μm in Enlargement. (G and I) Quantification of puncta ratio of yellow vs. red ( n = 10). (J) Enzymatic reaction kinetics of PP2A with different concentrations of pThr peptide. PP2A was isolated from HEK293 cells with the treatment of a PP2A inhibitor OA, FLAG-PPP2R5C, or siPPP2R5C ( n = 6). (K) Western blot confirms the changes in PPP2R5C levels. All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗∗ p < 0.01, ∗∗∗ p < 0.001, and n.s., no statistics, Student’s t test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: De-Phosphorylation Assay, Activity Assay, Western Blot, Derivative Assay, Infection, Fluorescence, Transfection, Marker, Plasmid Preparation, Isolation

Journal: Cell Reports Medicine

Article Title: Neuronal PPP2R5C in plasma is a potential biomarker for early diagnosis of Alzheimer’s disease

doi: 10.1016/j.xcrm.2026.102631

Figure Lengend Snippet: PPP2R5C triggers autophagy through ULK1-PPP2R5C direct binding activation (A) Western blot showing p -ULK1 Ser556 negatively correlated with PPP2R5C expression. (B) Binding positions and interaction mode analysis of proteins ULK1 and PPP2R5C. The binding interface was shown as a surface, and each protein was shown as a cartoon (right). The detailed molecular interactions of salt bridges, hydrogen bonds, and hydrophobic interactions were shown in the enlarged image, and residues on the protein interface are shown as sticks. (C) Co-immunoprecipitation assay to detect the interaction between PPP2R5C and ULK1 in HEK293 cells with FLAG-PPP2R5C or FLAG vector transfection. The co-precipitated ULK1 was subsequently detected by western blot analysis. (D) PPP2R5C and ULK1 interaction determined by co-immunoprecipitation and western blot in the cortex of WT and Tau P301S. (E) Immunofluorescence co-localization was used to observe the spatial location of PPP2R5C and ULK1 in the TauP301S and their WT littermates. Scale bars: 50 μm in normal and 20 μm in Enlargement. (F) Quantification of colocalization in (E) ( n = 5). (G) Mechanism diagram. In the early stages of AD, PPP2R5C protein levels begin to decrease. As a PP2A enzyme catalytic subunit B family member, PPP2R5C plays a role in AD pathology by influencing Tau protein phosphorylation. Additionally, the reduction of PPP2R5C affects the ULK1-PPP2R5C-autophagy pathway, resulting in elevated total Tau (t-Tau) protein levels, further contributing to AD progression. All the western blot data are representative of three independent experiments. Quantification data are expressed as mean ± SEM (∗∗ p < 0.01, Student’s t test).

Article Snippet: The Flag-PPP2R5C plasmid was modified from Flag/ZsGreen-PPP2R5C, siRNA PPP2R5C was purchased from Santa Cruz Biotechnology (#sc-45847).

Techniques: Binding Assay, Activation Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Phospho-proteomics