Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1
Bibliographic record
Abstract
Intramolecular interaction is a common mechanism that regulates protein activities.Conventionally, such interactions are investigated by classical in vitro biochemical assays.Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation assay (PLA).PLA is a specific and sensitive method that allows the observation of interacting proteins by target-specific antibody detection coupled to rolling circle amplification.ELMO1 is the regulatory subunit of ELMO1-dedicator of cytokinesis 180 (DOCK180) bipartite Rac1 guanine nucleotide exchange factor (GEF) which adopts a closed autoinhibitory conformation via an intramolecular interaction of its N-terminal ELMO inhibitory domain (EID) and Cterminal ELMO autoregulatory domain (EAD).In the assay, PLA signals are detected in cells transfected with ELMO1 1-315 and ELMO1 315-727 fragments.Moreover, overexpression of FE65, a neuronal adaptor which has been shown to disrupt ELMO1 intramolecular interaction, reduces the PLA signals of the two ELMO1 fragments significantly.Together, our results demonstrate that PLA can be employed for studying protein intramolecular interaction.
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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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| 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 | 0.003 |
| Insufficient payload (model declined to judge) | 0.007 | 0.002 |
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".