DNA-PK Activity Detection

DNA-PK activity was measured using AQT0440 selective sensor peptide (15 µM) with crude lysates (2 µg/well) from multiple cancer-derived cell lines with or without treatment.

Treatment with Neocarzinostatin or Cobalt chloride, which induce DNA double-strand breaks, resulted in robust activation of DNA-PK—up to 10-fold and 14-fold increases, respectively, across diverse models.

Lysates from HCT116 colon cancer cells treated with Neocarzinostatin showed the highest activity, with a signal almost 2-fold greater than any other cell line. This lysate was used for further validation studies.

• DNA-PK activity in Neocarzinostatin-treated HCT116 lysates was linear across 0.078–5.0 µg/well, spanning a 64-fold dynamic range.
• The signal was selectively inhibited (97% block) by the DNA-PK inhibitor AZD-7648 (1 µM).
• An IC₅₀ of 6.5 nM was determined for AZD-7648 in untreated HCT116 lysates.
• The sensor peptide substrate AQT0440 exhibited an apparent Km of 31 µM with Neocarzinostatin-treated HCT116 lysates.

These results confirm that PhosphoSens detects true DNA-PK catalytic activity in complex lysates, not indirect phosphorylation readouts or artifacts.

JNK1/2/3 Activity Detection

JNK activity was measured using AQT1196 selective sensor peptide (15 µM) with crude lysates from HEK293 and HeLa cells ± anisomycin stimulation and the selective JNK inhibitor JNK-IN-8 (1 µM).

• Anisomycin stimulation produced a vigorous induction of JNK activity, with a 49-fold increase in HEK293 and a 20-fold increase in HeLa lysates.
• Activity in anisomycin-treated HEK293 lysates was linear from 0.3–16 µg/well, spanning a 53-fold dynamic range.
• The AQT1196 sensor peptide exhibited a Km of 6.5 µM with anisomycin-treated HEK293 lysates.
• 97% of the signal was blocked by the JNK1/2/3 inhibitor JNK-IN-8 (1 µM), confirming the assay measures bona fide JNK catalytic activity.
• An IC₅₀ of 533 nM was determined for JNK-IN-8 in anisomycin-treated HEK293 lysates with AQT1196.

These studies highlight robust, stimulus-dependent JNK activation in lysates and demonstrate selective inhibition. Importantly, the magnitude of activation varies with cell type, serum conditions, and stimulus strength/duration, providing flexibility to optimize JNK activity detection. While total JNK protein levels can be measured by WB or ELISA, they are not expected to change under short stimulation windows—making PhosphoSens a more functional, activity-based readout.

AKT1/2/3 Activity Detection

AKT activity was measured using AQT0982 selective sensor peptide (10 µM) with crude lysates (2 µg/well) from multiple cell lines with or without growth factor stimulation.

• NIH3T3 cells treated with PDGF-BB (50 ng/mL) showed a robust 3-fold increase in AKT activity compared to unstimulated controls.
• Assay signal with PDGF-stimulated NIH3T3 lysates was linear from 0.078–1.5 µg/well, spanning a 19-fold dynamic range.
• The sensor peptide substrate AQT0982 had an apparent Km of 16 µM with PDGF-stimulated NIH3T3 lysates.
• Complete inhibition of AKT activity was observed with 1 µM Rizavasertib (ATP-competitive) or 1 µM Vevorisertib (allosteric) inhibitors.
• Inhibition was highly potent, with IC₅₀ values of 11 nM (Rizavasertib), 42 nM (Vevorisertib), and 5.2 nM when used in combination.

Cell lysate panel results: In four cell lines, AKT activity was stimulated by treatment, with the strongest effect in NIH3T3 cells ± PDGF (up to 8-fold stimulation). Incorporation of selective inhibitors blocked the signal completely, confirming that PhosphoSens detects true AKT catalytic activity in complex lysates.

ERK1/2 Activity Detection

ERK1/2 activity was measured using AQT1076 selective sensor peptide (15 µM) with lysates (1–5 µg/well) from seven cell lines ± PDGF stimulation and with/without ERK1/2 inhibitors.

• NIH3T3 cells (serum-deprived, then PDGF-stimulated) exhibited a robust 10-fold increase in ERK1/2 activity compared to unstimulated controls.
• In contrast, other cancer-derived cell lines showed constitutive ERK1/2 activity that was not further stimulated by PDGF.
• Incorporation of ERK1/2-selective inhibitors SCH772984 or Vx-11e (1 µM) completely blocked activity, confirming the signal as bona fide ERK1/2 catalytic activity.
• NIH3T3 + PDGF lysates showed 92-fold higher activity than unstimulated controls.
• The assay was linear from 0.31–10 µg/well, spanning a 32-fold range—much broader than recombinant ERK1/2 (4-fold).
• The AQT1076 substrate had an apparent Km of 17 µM.
• IC₅₀ values for inhibitors were 9.3 nM (SCH772984) and 101 nM (Vx-11e).

Validation & Context: Dual phosphorylation of ERK1/2 was also demonstrated by western blotting, consistent with PhosphoSens results, though WB is only semi-quantitative and limited by the complexity of ERK1/2’s >20 regulatory phosphorylation sites. The PhosphoSens Lysate Assay, in contrast, provides a direct, functional, and highly quantitative measure of ERK1/2 activity in its full cellular context.

GSK3A/B Activity Detection

GSK3A/B activity was measured using AQT1211 selective sensor peptide (15 µM) with crude lysates (2 µg/well) from multiple cell lines ± treatment and with/without the GSK3 inhibitor LY2090314 (1 µM).

• In two models, treatment reduced GSK3 activity by >3-fold (green bars), consistent with AKT-mediated phosphorylation and inactivation of GSK3.
• In A375 cells, GSK3 activity was measured at 19 RFU/min under basal conditions. This signal can likely be further optimized with alternative treatment conditions.
• GSK3A/B activity was linear from 0.078–1.3 µg/well, spanning a 17-fold dynamic range.
• The AQT1211 sensor peptide substrate had a Km of 5.1 µM with A375 lysates.
• Incorporation of the GSK3-selective inhibitor LY2090314 (1 µM) completely blocked GSK3A/B activity, confirming assay selectivity.
• The IC₅₀ value for LY2090314 was 3.3 nM with lysates from A375 cells.

Conclusion: The PhosphoSens Lysate Assay for GSK3A/B provides a direct, selective, and highly quantitative measure of GSK3 activity in complex cellular environments, distinguishing true kinase function from indirect phospho-protein readouts.

p38 Activity Detection

P38 activity was measured using the AQT1280 selective sensor peptide (15 µM) with crude lysates (2 µg/well) from eight cell models ± anisomycin stimulation and with/without the selective P38 inhibitor Ralimetinib (1 µM). The ERK1/2 inhibitor SCH772984 (1 µM) was included to ensure assay selectivity given potential ERK1/2 cross-reactivity in certain cell types.

• In multiple cell models, anisomycin treatment induced robust P38 activation ranging from 2- to 48-fold (green bars).
• HeLa cells (1 µM anisomycin, 15 min) exhibited the highest P38 induction (~12.5-fold), while MCF7, U87MG, MiaPaca-2, and Calu-6 cells showed 48-, 17-, 23-, and 17-fold increases, respectively.
• Incorporation of the selective P38 inhibitor Ralimetinib (1 µM) completely blocked signal, confirming assay selectivity.
• Inclusion of the ERK1/2 inhibitor SCH772984 (1 µM) further ensured P38-specific activity in models with elevated ERK1/2 expression.

Conclusion: The PhosphoSens Lysate Assay for P38 provides a direct, selective, and highly quantitative measure of endogenous P38 kinase activity across diverse cellular backgrounds, enabling detection of stress-induced signaling with high sensitivity.

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