αPIX Is a Trafficking Regulator that Balances Recycling and Degradation of the Epidermal Growth Factor Receptor.
Part of the results reproduced; minor but material deviations remained.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓No authors-side cause for any deviation
- ✓Any deviation was negligible
- 🔴Could not use the authors’ exact input data
- 🔴Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
▸Reproduction agent’s raw note
DROP (non_pipeline) — INDEPENDENTLY RE-CONFIRMED on 2026-06-22 against the live PLoS ONE article (this room was re-queued; the prior reproduction/ folder was archived to _requeue_archive/20260622T112805Z/). Paper: 'alphaPIX Is a Trafficking Regulator that Balances Recycling and Degradation of the Epidermal Growth Factor Receptor', Kortuem F, Harms FL, Hennighausen N, Rosenberger G., PLoS ONE 2015, DOI 10.1371/journal.pone.0132737, PMCID PMC4503440. I fetched the journal article and reviewed its full Materials & Methods: plasmid construction / PCR mutagenesis / Gateway cloning, Flp-In stable cell lines, transient transfection, siRNA knockdown of alphaPIX (ARHGEF6), co-immunoprecipitation, immunoblotting/Western, immunofluorescence + confocal microscopy, cell-surface biotinylation pulse-chase EGFR trafficking assays (degradation with primaquine; recycling with leupeptin/pepstatin A), EGFR ubiquitination assay, BrdU proliferation. The only computational elements are ImageJ densitometry of blots and Student's t-tests — NOT a bioinformatic/omics pipeline. NO high-throughput / sequencing / microarray / mass-spectrometry / bioinformatics analysis is described. Data Availability (verbatim): 'All relevant data are within the paper and its Supporting Information files.' NO deposited data accession (GEO/SRA/ENA/ArrayExpress/PRIDE/figshare/Zenodo/Dryad) and NO analysis code repository exists. Every reported quantitative value is a densitometric immunoblot/trafficking-assay readout (wet-lab), not a pipeline output, so nothing is pipeline-derivable to regenerate and nothing is available to profile -> empty claims[] and datasets[]. No «our HPC» compute warranted. This is a well-founded, controlled drop per BRIEF rules 2 & 6 and the SCREENING non_pipeline vocabulary. NOT attempted: any wet-lab result (out of scope by design).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessmentassessed: 2026-06-19 ⛓ 19be7120d0d7
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-22
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether and how the Rho-GEF αPIX, via its interaction with the E3 ubiquitin ligase c-Cbl, regulates endocytic sorting (degradation versus recycling) of the epidermal growth factor receptor (EGFR).
- ★ αPIX interacts with c-Cbl, including as endogenous proteins finding
- ★ EGF stimulation induces αPIX::c-Cbl complex formation, and this complex formation together with c-Cbl ligase activity is required for EGF-induced degradation of both proteins finding
- ★ αPIX sequesters c-Cbl away from EGFR, reducing EGFR ubiquitination and lysosomal degradation mechanism
- ★ αPIX strongly promotes EGFR recycling to the cell surface via its GIT-binding domain, independent of c-Cbl binding or αPIX GEF exchange activity finding
- ★ Trp197 in the αPIX SH3 domain and Arg829 in the c-Cbl PKPFPR motif are essential residues for the αPIX::c-Cbl interaction finding
- Both proteasomal (MG132) and lysosomal (chloroquine) inhibition stabilize EGF-induced αPIX and c-Cbl protein levels finding
- αPIX overexpression alters the number and morphology of EEA1-positive early endosomes following EGF stimulation finding
- CHO cells, which express few endogenous EGFRs but retain full endocytic machinery, are a suitable model for studying ectopically expressed EGFR trafficking method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| co-immunoprecipitation / western blot | CHO-K1 cells (endogenous proteins) | none (basal 10% FBS culture) | co-precipitation of αPIX with c-Cbl | — |
| co-immunoprecipitation / western blot | COS-7 cells, transfected with αPIX/c-Cbl domain-deletion and point mutants (ΔSH3, W197K, ΔGBD, ΔCC, R829A, C381A) | overexpression of mutant constructs | co-precipitation of HA-αPIX with c-Cbl variants | anti-HA-conjugated agarose beads, SDS-PAGE/immunoblot |
| time-course co-immunoprecipitation with densitometric quantification | COS-7 cells co-expressing HA-αPIX WT and c-Cbl WT | EGF stimulation (5 ng/ml, 5-60 min) after serum starvation, vs. +10% FBS | relative amount of c-Cbl co-precipitated with αPIX; αPIX and c-Cbl total protein levels | immunoblot/autoradiographic densitometry |
| protein stability assay with proteasomal/lysosomal inhibitors | COS-7 cells co-expressing HA-αPIX WT and c-Cbl WT | MG132 (20 μM) or chloroquine (50 μM), 6h, plus EGF (25 ng/ml) | αPIX and c-Cbl protein levels | immunoblot |
| surface biotinylation pulse-chase EGFR trafficking assay | CHO cell lines stably expressing V5-αPIX WT/W197K/GEF-/ΔGBD or CAT (control), transiently expressing EGFR | EGF stimulation (25 ng/ml, 30 min pulse) then chase | intracellular biotinylated EGFR levels over chase time | autoradiography/immunoblot |
| immunofluorescence confocal microscopy | COS-7 cells transiently expressing HA-αPIX WT (endogenous EGFR) | EGF pulse (25 ng/ml, 30 min) and 30 min chase | number/morphology of EEA1-positive early endosomal vesicles | Alexa Fluor 488/546 immunofluorescence, DAPI |
| surface biotinylation pulse-chase with recycling blockade | CHO cell lines stably expressing αPIX variants, transfected with EGFR | EGF (30 min pulse) plus primaquine (0.3 mM) to block recycling | intracellular EGFR levels over chase time (degradation kinetics) | immunoblot/autoradiography |
| rescue biotinylation degradation assay | αPIX WT-expressing CHO cells co-transfected with c-Cbl | c-Cbl overexpression rescue | restoration of EGF-induced EGFR degradation | immunoblot |
- – Endogenous αPIX (87 kDa) co-precipitates with c-Cbl in CHO-K1 cells under basal conditions
- ▼ αPIX ΔSH3 and αPIX W197K show drastically diminished co-IP with c-Cbl WT, while ΔGBD and ΔCC still bind
- – c-Cbl R829A abolishes interaction with αPIX WT, but c-Cbl C381A (ligase-dead) still binds αPIX
- ▲ Co-precipitated c-Cbl with αPIX gradually increases up to 30 min of EGF stimulation, strongest under +10% FBS
- – αPIX W197K or c-Cbl R829A expression stabilizes αPIX/c-Cbl protein levels upon EGF stimulation, blocking their normal EGF-induced decrease
- – Both MG132 and chloroquine maintain αPIX and c-Cbl protein levels after EGF stimulation
- ▲ Intracellular EGFR accumulates after 30 min chase in αPIX WT-expressing CHO cells but not in CAT control cells
- – In primaquine-blocked assays, intracellular EGFR remains constant in αPIX WT, GEF-, and ΔGBD cells but decreases in control and αPIX W197K cells; c-Cbl co-expression rescues degradation in αPIX WT cells
- other 87 kDa (molecular weight of co-precipitated endogenous αPIX with c-Cbl in CHO-K1 cells)
- count n = 6 independent experiments (quantification of c-Cbl co-precipitated with HA-αPIX before/after EGF (Fig 2A), paired t-test)
- count n = 5 independent experiments (quantification of HA-αPIX WT and c-Cbl WT levels after EGF stimulation (Fig 2B), paired t-test)
- count n = 4 independent experiments (quantification of αPIX/c-Cbl levels with MG132/chloroquine treatment (Fig 2C), unpaired t-test)
- count n = 4 independent experiments (quantification of intracellular EGFR fractions in trafficking assay (Fig 3B), unpaired t-test)
- other Trp196/Trp197 (αPIX SH3 domain); Arg829 (c-Cbl PKPFPR motif) (residues critical for αPIX::c-Cbl interaction)
- other EGF 5 ng/ml or 25 ng/ml (stimulation doses used across complex-formation and trafficking assays)
- other MG132 20 μM; chloroquine 50 μM; primaquine 0.3 mM (inhibitor concentrations used in degradation/recycling-block assays)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper uses cell-based biochemical assays (co-immunoprecipitation, surface biotinylation pulse-chase, and immunofluorescence microscopy) in CHO-K1, COS-7, and stable CHO cell lines to characterize αPIX/c-Cbl interaction and EGFR trafficking. Quantitative comparisons are based on densitometric quantification of immunoblot/autoradiographic signals across multiple independent experiments (n = 4–6), summarized as mean ± SD, with paired or unpaired Student's t-tests used to compare conditions (e.g., unstimulated vs. EGF-stimulated, or different cell lines/genotypes at a given timepoint). Immunofluorescence data (Fig 3C) are presented as representative images from examined cells without an accompanying statistical test.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Paired Student's t-test | Fig 2A: quantification of c-Cbl co-precipitated with HA-αPIX, unstimulated vs. 30 min EGF | n = 6 independent experiments | not stated |
| Paired Student's t-test | Fig 2B: relative HA-αPIX and c-Cbl protein levels, unstimulated vs. EGF-stimulated timepoints | n = 5 independent experiments | not stated |
| Unpaired Student's t-test | Fig 2C: relative HA-αPIX/c-Cbl levels with MG132 or chloroquine vs. vehicle | n = 4 independent experiments | not stated |
| Unpaired Student's t-test | Fig 3B: relative amounts of intracellular EGFR, αPIX WT vs. control (CAT) cell lines over chase time | n = 4 independent experiments | not stated |
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Multiple independent t-tests are used across several figures and timepoints/genotypes (Fig 2A, 2B, 2C, 3B) without a stated correction for multiple comparisons.↳ Could also: A one-way or two-way ANOVA with a post-hoc test (e.g., Tukey or Dunnett) applied within each experiment's family of comparisons — This would jointly model all groups/timepoints at once and control the family-wise error rate across the multiple comparisons being made, which is a standard approach when more than two groups or conditions are compared.
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Quantitative comparisons rely on Student's t-tests with relatively small sample sizes (n = 4–6 independent experiments).↳ Could also: A non-parametric alternative such as the Wilcoxon signed-rank test (for paired data) or Mann-Whitney U test (for unpaired data) — Non-parametric tests do not rely on an assumption of normally distributed differences, which can be difficult to verify with small n, and can serve as a complementary or more conservative check on the parametric result.
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Variability is summarized using the standard deviation (± sd) in the quantification graphs.↳ Could also: Reporting the standard error of the mean (SEM) or a 95% confidence interval alongside or instead of SD — A CI directly conveys the precision of the estimated mean and the plausible range of the true effect, which can be particularly informative when n is small.
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Quantification of protein levels and EGFR trafficking is based on densitometric analysis of immunoblot/autoradiographic signals from repeated independent experiments, each compared with a single t-test per timepoint/condition.↳ Could also: A mixed-effects (repeated-measures) model treating experiment/blot as a random effect and condition/timepoint as a fixed effect — This approach can jointly account for experiment-to-experiment variability and multiple timepoints within the same experiment, potentially increasing statistical power and providing effect estimates across the full time course rather than pairwise snapshots.
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Immunofluorescence quantification of EEA1-positive vesicle number/morphology (Fig 3C) is presented as representative images of examined cells without a reported statistical test.↳ Could also: Formal quantitative image analysis (e.g., counting vesicle number/size per cell) summarized with descriptive statistics and compared using a t-test, Mann-Whitney U test, or mixed-effects model across the examined cells — Adding a quantitative statistical comparison alongside representative images can provide an objective, reproducible measure of the observed morphological difference in addition to the qualitative visual example.
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Figure legends indicate that P values were calculated but the specific numeric values, exact test assumptions, and software/tool used for the calculations are not detailed in the text.↳ Could also: Reporting exact p-values (rather than threshold-based significance), the software/package used, and confirmation of normality or variance-equality assumptions — This level of detail allows readers to fully evaluate the strength of evidence and reproduce the statistical analysis independently.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-26177020
Title: αPIX Is a Trafficking Regulator that Balances Recycling and Degradation of the Epidermal Growth Factor Receptor. Authors: Kortüm F, Harms FL, Hennighausen N, Rosenberger G. Journal: PLoS ONE (2015) · DOI 10.1371/journal.pone.0132737 · PMCID PMC4503440
Verdict: OUT OF SCOPE — non_pipeline (DROP)
This is a classical wet-lab cell-biology study. There are no pipeline-derived computational results to reproduce and no deposited datasets to profile.
What the paper actually contains (all wet-lab / manual)
- Co-immunoprecipitation & GST pull-down (αPIX ⇄ c-Cbl ⇄ EGFR interactions)
- Cell-surface biotinylation pulse-chase trafficking assays (EGFR recycling vs. degradation), quantified by densitometry of immunoblots
- Western blotting / immunoblotting + densitometric quantification
- Immunofluorescence confocal microscopy (EGFR / endosomal co-localization)
- siRNA knockdown of αPIX (ARHGEF6)
- In-vivo ubiquitination assays (HA-ubiquitin)
- BrdU proliferation assay
- Cell lines: CHO-K1, COS-7, CHO
Why it is non-reproducible in this study's sense
| Requirement for an in-scope reproduction | Present? | Evidence |
|---|---|---|
| Bioinformatic pipeline producing a reported value | No | Methods describe only bench assays; no sequencing/array/MS/imaging-pipeline analysis |
| Public deposited data accession (GEO/SRA/ENA/PRIDE/figshare/Zenodo/Dryad) | No | Data Availability: "All relevant data are within the paper and its Supporting Information files." |
| Analysis code / software repository | No | None cited in paper or PLOS metadata |
| Identifiable pipeline-derived reported value to compare against | No | All reported numbers are densitometric quantifications of gels/blots — wet-lab readouts, not pipeline outputs |
Per BRIEF HARD RULE 2 + 6 and SCREENING drop vocabulary, the correct controlled
outcome is non_pipeline (text-mining false positive: the publication has no
computational pipeline to reproduce). This is a valid outcome and does not
require «our HPC» compute. No accession exists, so dataset_profile.json carries no
entries (none to profile).
No individual results have been recorded for this entry yet.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a correctly determined non_pipeline DROP: the αPIX/EGFR trafficking study is entirely wet-lab cell biology (co-IP, pull-down, biotinylation pulse-chase, densitometry, IF, siRNA) with no code repository and no deposited data accession (PLOS 'data within the paper/SI'). Every reported number is a manual densitometric quantification, not a pipeline output, so nothing could be regenerated and no value could be put 1:1 against a reproduced result. The drop sits on data availability / study type (our scope), not an authors' defect — there is no fabrication signal, so q5/q7/q8 are yellow (not assessable) rather than red.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.