pp1 inhibitor okadaic acid  (MedChemExpress)

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

Journal: Cells

Article Title: Midbody Proteins Display Distinct Dynamics during Cytokinesis

doi: 10.3390/cells11213337

Figure Lengend Snippet: Different expression profiles of midbody proteins during mitotic exit. ( A ) Time course analysis of midbody protein expression during mitotic exit. HeLa cells were synchronized by thymidine/nocodazole block and then collected at the indicate time points after nocodazole release. Proteins were extracted and used in Western blot analysis to identify the antigens indicated to the right. The numbers on the left indicate the sizes of the molecular mass marker. ( B ) Graph showing the quantification of protein levels, normalized to tubulin and relative to levels at time 0 min, from at least two different Western blots like the one shown in ( A ) using protein extracts from two separate experiments. ( C ) Effect of different inhibitors on midbody protein levels. HeLa cells were synchronized by thymidine/nocodazole block, released for 45 min in fresh medium, and then incubated for further 60 min in MG132, okadaic acid (OKA), tautomycetin (TAUT), ZM447439 (ZM), or the solvent DMSO as control. Proteins were extracted and used in Western blot analysis to identify the antigens indicated to the right. The numbers on the left indicate the sizes of the molecular mass marker. ( D ) Graph showing the quantification of protein levels, normalized to tubulin and relative to DMSO levels, from at least two different Western blots like the one shown in ( C ) using protein extracts from two separate experiments.

Article Snippet: Mitotic cells were washed five times with PBS, and released in fresh medium containing either one of the following drugs: 10 μM MG132 (proteasome inhibitor, Sigma), 50 nM okadaic acid (PP1 and PP2A inhibitor, Calbiochem, San Diego, CA, USA), 2 μM tautomycetin (PP1 inhibitor, TOCRIS, Bristol, UK), 2 μM ZM447439 (AURKB inhibitor, TOCRIS) or the DMSO solvent as control.

Techniques: Expressing, Blocking Assay, Western Blot, Marker, Incubation, Solvent, Control