okadaic acid  (Cayman Chemical)

Journal: Acta Neuropathologica Communications

Article Title: 4R-tau isoform induction via TDP-43 in neurons in response to insulin: converging signaling pathways with implications for neurodegenerative disease

doi: 10.1186/s40478-025-02174-x

Figure Lengend Snippet: Inhibition of CK1 reduces insulin-induced 4R-tau expression and TDP-43 phosphorylation in cortical neurons. A Representative Western blot (WB) of cortical neuron lysates pretreated for 1 h with kinase and phosphatase inhibitors prior to insulin (Ins) stimulation. Inhibitors included D4476 (CK1 inhibitor), DMAT (CK2 inhibitor), and okadaic acid (OA; PP1a/PP2a inhibitor), each tested at two concentrations (×1 and ×10). Detection with a 4R-tau –specific antibody reveals a marked reduction in the insulin-induced 4R-tau signal only in D4476 + Ins-treated neurons, with both concentrations showing strong inhibition. D4476 at ×1 significantly reduces 4R tau induction. B WB showing electrophoretic mobility of TDP-43 under the same treatment conditions as in (A). The mobility shift observed in Ins- and OA-treated samples (consistent with phosphorylation) is absent in D4476 + Ins-treated neurons, supporting CK1 involvement in TDP-43 phosphorylation. C Schematic of CK inhibitors used: D4476 selectively inhibits CK1, and DMAT inhibits CK2, both kinases implicated in TDP-43 phosphorylation D Representative WB showing the effect of different pre-incubation times (1, 2, and 4 h) with D4476 (×1) prior to insulin stimulation. A time-dependent decrease in 4R-tau signal is observed, indicating progressive inhibition of insulin-induced expression. Right: Quantification of 4R-tau levels normalized to tubulin from three independent experiments. Insulin treatment significantly increases 4R-tau expression compared to control, and this induction is progressively inhibited by D4476 pretreatment (**** p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test). No 4R-tau signal was detected in untreated (Ctr) or in the 4 h D4476 + Ins condition (nd). E Representative membranes showing 4R-tau (upper panel) and TDP-43 (lower panel) expression in control (Ctr), Ins, and D4476 (4 h pre-incubation) + Ins conditions. While 4R-tau levels increase upon insulin stimulation and are reduced by D4476, TDP-43 levels remain unchanged across conditions

Article Snippet: ; mouse NGF (nerve growth factor 2.5 S Alomone N-100 ) at 100 ng/ml for 60 min as in [ ]; Okadaic Acid ( OA ) as PP1a and PP2a 10 nM and 100 nM 24 h before incubation with insulin as used in [ ] ; CK1 inhibitor D4776 (Medchemexpress, stock 1 mg/25 ml DMSO) 100 μM and 1000 μM 1 h before other treatment was used at the following concentrations before incubation with insulin as used in [ ]; CK2 inhibitor DMAT (Medchemexpress, HY-15535, (stock 1 mg/DMSO) 20 μM and 200 μM, 24 h incubation before other treatment, as previously used in [ ].

Techniques: Inhibition, Expressing, Phospho-proteomics, Western Blot, Mobility Shift, Incubation, Control

Journal: Nature Communications

Article Title: CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on attachments

doi: 10.1038/s41467-025-64148-w

Figure Lengend Snippet: a Experimental scheme to test factors that regulate polar chromosome congression following washout of a CENP-E inhibitor. b Percentage of polar chromosomes per cell that successfully aligned within 30 min of washout under the indicated treatments. Colored points represent individual cells; black lines show the mean, with light and dark gray areas marking 95% confidence intervals for the mean and standard deviation, respectively. Numbers: 23, 4, 13, 24, 11, 23, 21, 18, 10 cells, all from ≥3 independent biological replicates. Representative images of CENP-A-GFP and centrin1-GFP expressing RPE-1 cells (centrioles circled) over time after washout of CENP-E inhibitor GSK-923295 in phosphatase-pre-inhibited (Okadaic acid) ( c ), Aurora B acutely inhibited (ZM447439) ( d ), Spindly-depleted ( e ) and BuBR1-depleted ( f ) cells. Images are maximum projections color-coded by depth (color bar in Fig. ). Arrowheads mark polar kinetochores failing to congress. Statistics: two-tailed ANOVA with Tukey’s HSD post hoc test. Symbols: ****, P ≤ 0.0001; inh., inhibited; depl., depleted; siRNA, small interfering RNA; PP, protein phosphatases; KK, Kid and Kif4a; chrom., chromosome. Source data are provided as a Source Data file.

Article Snippet: Inhibitor of PP1 and PP2A Okadaic acid (MedChemExpress, IC 50 value 0.1–0.3 nM for PP2A and 15–50 nM for PP1) at a final concentration of 1 μM, was added acutely before imaging or 30 min before washout of the CENP-E inhibitor as noted.

Techniques: Standard Deviation, Expressing, Two Tailed Test, Small Interfering RNA

Journal: Nature Communications

Article Title: CENP-E initiates chromosome congression by opposing Aurora kinases to promote end-on attachments

doi: 10.1038/s41467-025-64148-w

Figure Lengend Snippet: a Aurora kinases inhibit congression initiation by phosphorylating the Ndc80 tail near centrosomes (1). On polar kinetochores, CENP-E–BubR1 facilitates early end-on attachment formation near the Aurora A gradient (2), triggering a decline in Aurora B activity, loss of Mad2 from the kinetochore, and stabilization of Ndc80-microtubule binding, all preceding fast kinetochore movement (3). b This establishes a negative feedback loop involving Aurora B, CENP-E, BubR1-PP2A, the fibrous corona, and outer kinetochore proteins (Knl1 and Hec1/Ndc80). The loop self-limits by promoting end-on attachment and initiating chromosome congression, which in turn reduces the upstream signals that sustain it. Lines with arrows indicate activation, and blunt lines indicate deactivation by phosphorylation or direct physical inhibition. Initially, polar chromosomes form lateral kinetochore-microtubule attachments that fail to convert to stable end-on attachments without CENP-E due to high Aurora B activity. Aurora B phosphorylates Knl1 and Hec1 to prevent end-on conversion and maintains the expanded fibrous corona (depicted as a crown), which further inhibits attachment stabilization. Aurora B also activates CENP-E via phosphorylation. We propose (indicated by a question mark) that activated CENP-E interacts with BubR1 and possibly PP2A to counteract Aurora B-mediated phosphorylation, enabling stabilization of initial end-on attachments, progressive corona disassembly, and chromosome congression toward the spindle midplane.

Article Snippet: Inhibitor of PP1 and PP2A Okadaic acid (MedChemExpress, IC 50 value 0.1–0.3 nM for PP2A and 15–50 nM for PP1) at a final concentration of 1 μM, was added acutely before imaging or 30 min before washout of the CENP-E inhibitor as noted.

Techniques: Activity Assay, Binding Assay, Activation Assay, Phospho-proteomics, Inhibition