The importance of complete tissue homogenization for accurate stoichiometric measurement of myosin light chain phosphorylation in airway smooth muscle
Bibliographic record
Abstract
The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea-glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10(-6) mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.003 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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 itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".