In EGF-stimulated HeLa cells, PD184352 suppressed ERK1/2 phosphorylation at 0.5 µM after a 1 h pretreatment. Higher concentrations also affected the ERK5 mobility response. These signaling results support concentration-dependent pathway inhibition and do not establish a cell-killing effect. [1]
Inhibition of active MKK1 in a kinase selectivity panel
Biochemical assay · Purified active phosphorylated MKK1 (MEK1), with a broader protein kinase comparison panel [1]
The authors describe an IC50 close to 1 µM against active phosphorylated MKK1. Table 3 reports 12±1 percent MKK1 activity remaining at 10 µM PD 184352 relative to inhibitor-omitted controls. This assay result is distinct from potency against pathway activation in intact cells.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 µM for the Table 3 selectivity measurement; IC50 assessed separately across ten concentrations.
- Exposure duration
- 30 min kinase assay
- Method
- Radiometric kinase assay at 21 °C; MKK1 assayed at 5 µM ATP after activation with c-Raf.
- Reported limits
- Vehicle and the meaning of ± in Table 3 are not specified in the read assay methods/caption; no SEM or SD assignment made.
Methods and source locations
Compound reported in the paper: PD184352 (CI-1040); Compound label in the paper: PD 184352
Results concern the authors’ synthesized PD 184352. No supplier equivalence, salt conversion or Adooq lot validation is established.
Table 3, MKK1 row and caption; Materials and methods: Protein kinase inhibitors and other materials, Activation of protein kinases, Protein kinase assays; Results and Discussion: Inhibitors of MKK1 and its activation
Active phosphorylated MKK1 inhibition: Original Results and Discussion statement; no exact decimal inferred. — Results and Discussion: Inhibitors of MKK1 and its activation, second paragraph
MKK1 activity relative to inhibitor-omitted control: Table 3, PD 184352 (10 µM) column, MKK1 row. Caption: averages of duplicate determinations; experiments repeated two or three times with similar results. The ± statistic is not defined in that caption. — Table 3: header, MKK1 row and caption
Concentration-dependent ERK pathway suppression in EGF-stimulated HeLa cells
Cell-based study · Serum-starved HeLa cells stimulated with EGF [1]
PD 184352 suppressed EGF-induced ERK1/2 phosphorylation at 0.5 µM in the authors’ parallel inhibitor comparison. Figure 1 also shows that higher PD 184352 concentrations affect the ERK5 mobility response, limiting a blanket claim of ERK1/2-pathway selectivity at all concentrations. These immunoblots measure signaling rather than cell viability.
Literature-Reported Treatment Conditions
- Treatment concentration
- Figure 1 dose series: 0.001, 0.005, 0.025, 0.1, 0.5, 2.0, 10 and 20 µM PD 184352.
- Exposure duration
- 16 h serum starvation, followed by 1 h inhibitor incubation and 10 min EGF stimulation.
- Method
- EGF 100 ng/ml; immunoblotting for phosphorylated ERK1/2 and total ERK5; EGF-negative and EGF-positive inhibitor-absent lanes shown.
- Co-treatment
- EGF stimulation; other MEK inhibitors were tested in parallel, not combined with PD 184352.
- Reported limits
- Vehicle and biological replicate number are not given in the Figure 1 caption or read MKK1 section. Figure 1A does not display a total-ERK1/2 loading-control blot; no quantitative phosphorylation fraction is claimed.
Methods and source locations
Compound reported in the paper: PD184352 (CI-1040); Compound label in the paper: PD 184352
Results concern the authors’ synthesized PD 184352. No supplier equivalence, salt conversion or Adooq lot validation is established.
Figure 1A–B and caption; Results and Discussion: Inhibitors of MKK1 and its activation; Materials and methods: inhibitor synthesis and antibody sources
EGF-induced ERK1/2 phosphorylation: Phospho-ERK1/2 immunoblot; text states suppression at this concentration. Not an IC50 or a densitometric percentage. — Figure 1A and caption; Results and Discussion: Inhibitors of MKK1 and its activation, second paragraph
ERK5 electrophoretic mobility response to EGF: Total-ERK5 immunoblot mobility shift in Figure 1B, PD 184352 row; qualitative visual assessment only. — Figure 1B, PD 184352 row and caption
