Densitometric Analysis of eIF-4E Phosphorylation Data
| Treatments . | Mean Area* . | |||
|---|---|---|---|---|
| . | 30 min . | 1 h . | 2 h . | 4 h . |
| Control | 2.04 ± 1.1 | 2.93 ± 2.3 | 2.46 ± 1.2 | 3.02 ± 0.7 |
| GM-CSF | 5.80 ± 1.8† | 9.81 ± 2.3† | 4.36 ± 0.4† | 7.19 ± 3.1† |
| SLF | 6.10 ± 2.6† | 8.14 ± 3.4† | 5.26 ± 0.9† | 6.14 ± 2.7† |
| GM-CSF + SLF | 19.05 ± 2.1†‡ | 24.75 ± 5.1†‡ | 15.53 ± 2.1†‡ | 21.48 ± 4.6†‡ |
| MIP-1α + G + S | 7.87 ± 2.2ρ | 4.56 ± 0.7ρ | 3.92 ± 1.4ρ | 5.37 ± 1.2ρ |
| IP-10 + G + S | 8.39 ± 2.6ρ | 7.49 ± 1.3ρ | 4.48 ± 0.7ρ | 6.39 ± 2.3ρ |
| CT + G + S | 2.50 ± 0.8ρ | 5.38 ± 0.6ρ | 5.78 ± 1.2ρ | 8.39 ± 3.2ρ |
| PF4 + G + S | 18.17 ± 4.1 | 19.54 ± 2.1 | 15.82 ± 1.1 | 19.45 ± 0.9 |
| IL-8 + G + S | 21.08 ± 3.7 | 21.58 ± 4.5 | 13.71 ± 3.0 | 18.86 ± 2.6 |
| Treatments . | Mean Area* . | |||
|---|---|---|---|---|
| . | 30 min . | 1 h . | 2 h . | 4 h . |
| Control | 2.04 ± 1.1 | 2.93 ± 2.3 | 2.46 ± 1.2 | 3.02 ± 0.7 |
| GM-CSF | 5.80 ± 1.8† | 9.81 ± 2.3† | 4.36 ± 0.4† | 7.19 ± 3.1† |
| SLF | 6.10 ± 2.6† | 8.14 ± 3.4† | 5.26 ± 0.9† | 6.14 ± 2.7† |
| GM-CSF + SLF | 19.05 ± 2.1†‡ | 24.75 ± 5.1†‡ | 15.53 ± 2.1†‡ | 21.48 ± 4.6†‡ |
| MIP-1α + G + S | 7.87 ± 2.2ρ | 4.56 ± 0.7ρ | 3.92 ± 1.4ρ | 5.37 ± 1.2ρ |
| IP-10 + G + S | 8.39 ± 2.6ρ | 7.49 ± 1.3ρ | 4.48 ± 0.7ρ | 6.39 ± 2.3ρ |
| CT + G + S | 2.50 ± 0.8ρ | 5.38 ± 0.6ρ | 5.78 ± 1.2ρ | 8.39 ± 3.2ρ |
| PF4 + G + S | 18.17 ± 4.1 | 19.54 ± 2.1 | 15.82 ± 1.1 | 19.45 ± 0.9 |
| IL-8 + G + S | 21.08 ± 3.7 | 21.58 ± 4.5 | 13.71 ± 3.0 | 18.86 ± 2.6 |
Factor-starved MO7e cells maintained in phosphate-free medium and supplemented with [32P]orthophosphate were treated with growth factors for the times indicated or were pretreated with MIP-1α, IP-10, PF4, IL-8, or cholera toxin (CT) for 1 hour before combined treatment with GM-CSF and SLF for the times indicated. eIF-4E proteins immunoprecipitated from cell lysates were separated by 12% SDS-PAGE and blotted to PVDF membranes, as described in the Materials and Methods. Intensity of 32P-labeled proteins were visualized by autoradiography and differences between groups were determined by densitometry. eIF-4E protein content was determined by immunoblotting PVDF membranes with anti–eIF-4E antibodies and horseradish peroxidase-linked protein G. ECL-treated membranes were exposed to film, and autoradiograms were analyzed by densitometry.
Densitometric measurements for each group are expressed as the mean area under the peak, corresponding to band intensity on the autoradiogram, expressed as a percentage of the total area under the curve analyzed by the densitometer within each experiment. Values were normalized to protein content before statistical analysis. Each value represents the mean ± the standard deviation for five to six determinations within each treatment group obtained in separate experiments.
Significant difference from control mean (P < .05).
Significant difference from combined means of individual GM-CSF and SLF treatment groups within same treatment duration (P < .05).
ρ Significant difference from GM-CSF + SLF mean (P < .05).