In HEK-293-derived cells, 10 μM GSK429286A for 1 h reduced MYPT Thr850 phosphorylation under basal and constitutively active Rho conditions. ERM phosphorylation was not inhibited in the same extracts, showing that these downstream phosphorylation readouts responded differently. [1]
ROCK2 inhibition distinguished from LRRK2 inhibition
Biochemical assay · Recombinant His6–ROCK2-(2–543), GST–LRRK2 wild type and G2019S-(1326–2527) [1]
GSK429286A inhibited ROCK2 in a peptide phosphorylation assay. The Figure 4B table reports markedly weaker inhibition of both LRRK2 constructs. Values are assay endpoints, not cellular treatment concentrations.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response series shown in Figure 4B; not a treatment-dose recommendation.
- Exposure duration
- 15 min
- Method
- Peptide phosphorylation at 30 °C; activity relative to DMSO control.
- Vehicle
- DMSO, 0.1% of reaction volume
- Reported limits
- Specific peptide identity/concentration for Figure 4 is not explicit in the main figure caption; supplement unavailable. Caption states at least duplicate reactions, with similar results in at least one other experiment.
Methods and source locations
Compound reported in the paper: GSK429286A; Compound label in the paper: GSK429286A
Evidence applies to the named research compound; no supplier-lot equivalence or isolated-enantiomer claim.
Figure 4A–B; Results: Identification of selective small-molecule tool inhibitors of LRRK2; Materials and methods: LRRK2 and ROCK kinase assays
ROCK2 peptide phosphorylation: Radiometric peptide kinase assay; 100 μM ATP; nonlinear regression. — Figure 4B embedded IC50 table; LRRK2 and ROCK kinase assays
LRRK2 wild-type and G2019S peptide phosphorylation: Same figure assay; retain the bound printed in the embedded table. — Figure 4B embedded IC50 table
Kinase-panel limits on ROCK2 selectivity
Biochemical assay · 85-kinase profiling panel at ATP concentrations approximating each kinase ATP Km [1]
At 1 μM, GSK429286A strongly inhibited ROCK2 while leaving LRRK2 activity near control. The panel also showed inhibition of MSK1, RSK1 and S6K1, qualifying any claim of exclusive ROCK selectivity. Table values are residual activity, not IC50 values.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 μM
- Method
- Kinase panel compared with inhibitor-omitted control incubations.
- Reported limits
- Table 1 does not define the ± statistic or replicate count in its caption. Detailed profiling methods and kinase-specific ATP concentrations are assigned to the unavailable supplement; no values inferred.
Methods and source locations
Compound reported in the paper: GSK429286A; Compound label in the paper: GSK429286A
Evidence applies to the named research compound; no supplier-lot equivalence or isolated-enantiomer claim.
Table 1 and caption; Results: LRRK2 tool compound selectivity profiles; Other methods
ROCK2 residual activity: Kinase profiling at 1 μM GSK429286A; ATP approximately Km. — Table 1, GSK429286A (1 μM) column, ROCK2 row
LRRK2 residual activity: wild type; G2019S: Kinase profiling at 1 μM GSK429286A. — Table 1, GSK429286A column, LRRK2 rows
Residual activity: MSK1; RSK1; S6K1: Kinase profiling at 1 μM GSK429286A. — Table 1, GSK429286A column, MSK1/RSK1/S6K1 rows
MYPT phosphorylation inhibition with preserved ERM phosphorylation
Cell-based study · HEK-293-derived Flp-in T-REx cells, with or without doxycycline-induced GFP–G14V-Rho [1]
At 10 μM for 1 h, GSK429286A suppressed basal and G14V-Rho-associated MYPT Thr850 phosphorylation. ERM phosphorylation persisted in the same extracts. Total MYPT, ezrin, GFP and ERK blots support interpretation of the phosphoprotein readouts. No cellular potency or viability endpoint was measured here.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 μM GSK429286A
- Exposure duration
- 1 h; treatment began 7 h after induction where applicable
- Method
- Immunoblotting; G14V-Rho induction with 1 μg/ml doxycycline versus uninduced controls.
- Co-treatment
- No second inhibitor in the adopted GSK429286A lanes of Figure 6A; doxycycline used only for G14V-Rho induction.
- Reported limits
- Main Figure 6A caption does not specify vehicle concentration, biological replicate count or quantitative uncertainty. No densitometric percentages inferred.
Methods and source locations
Compound reported in the paper: GSK429286A; Compound label in the paper: GSK429286A
Evidence applies to the named research compound; no supplier-lot equivalence or isolated-enantiomer claim.
Figure 6A and caption; Results: Evaluation of ROCK and LRRK2 inhibitors in cells; Materials and methods: Antibodies and Immunological procedures
MYPT Thr850 phosphorylation: Phospho-MYPT and total MYPT immunoblot; untreated and other inhibitor comparisons. — Figure 6A, pMYPT T850 and MYPT rows; Results: Evaluation of ROCK and LRRK2 inhibitors in cells
ERM phosphorylation: Phospho-ERM immunoblot with ezrin/ERK comparisons in the same extracts. — Figure 6A, pERM, Ezrin and ERK rows
