Identification of a calcitriol-regulated Sp-1 site in the promoter of human CD14 using a combined western blotting elect rophoresis mobility shift assay (WEMSA)
Why this work is in the frame
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Bibliographic record
Abstract
Calcitriol (1alpha, 25-dihydroxyvitamin D(3)) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it