My Short-term Dbol-only Experiment Or, How I Learned To Stop Worrying And Love Oral Anabolics Part-1
How inhibiting myostatin (the "growth‑factor brake" on skeletal muscle) turns on thyroid‑hormone‑dependent growth pathways
Step What happens when the myostatin brake is removed How it feeds into the T3‑driven anabolic program
1. Loss of the myostatin–SMAD axis Myostatin binds to the activin IIB receptor → phosphorylates SMAD2/3 → they go to the nucleus and repress genes that encode growth‑promoting proteins (IGF‑1, myogenic transcription factors, etc.). Removing myostatin stops this repression. With SMAD signalling off, the cell’s "default" is a pro‑growth state: IGF‑1 rises, MyoD/MyoG increase, and the inhibitory brake on the mTORC1 pathway loosens.
2. Decrease in DACT/DAAM antagonists of Wnt SMADs normally up‑regulate DACT1 (a Wnt antagonist) and DAAM (modulates cytoskeletal dynamics). Without SMAD, these inhibitors fall. Less DACT/DAAM means canonical Wnt signalling can proceed unhindered; β‑catenin accumulates and translocates to the nucleus to activate target genes that cooperate with MyoD/MyoG.
3. Lowered expression of GSK‑3β SMAD positively regulates GSK‑3β, which phosphorylates β‑catenin marking it for degradation. Loss of SMAD reduces GSK‑3β levels. β‑catenin is less phosphorylated and thus stabilized, enhancing Wnt transcriptional output.
4. Decreased LRP6 phosphorylation LRP6 is the co‑receptor that must be phosphorylated to propagate the Wnt signal; SMAD up‑regulates its activation. With diminished SMAD activity, LRP6 phosphorylation drops. The Wnt pathway’s amplification step is weakened, lowering β‑catenin nuclear entry.
5. Reduced GSK3β phosphorylation (activation) Active GSK3β phosphorylates β‑catenin for degradation; its inactivation by phosphorylation is a key regulatory point. SMAD influences this switch. When SMAD is low, less GSK3β becomes phosphorylated/activated. β‑catenin remains more stable and available for signaling.
6-10. Various post‑translational modifications Phosphorylation at different residues can either target β‑catenin for degradation or stabilize it. SMAD signaling modulates the activity of kinases/phosphatases responsible for these events (e.g., CK1, GSK3β, PKC). The net effect depends on the balance: increased stability leads to enhanced signaling; otherwise, decreased signaling.
Bottom‑Line:
Higher phosphorylation at residues that target β‑catenin for degradation → Reduced protein levels and weaker downstream signaling.
Lower phosphorylation (or phosphorylation at stabilizing sites) → Accumulation of β‑catenin and stronger signaling.
3. How to Use This Knowledge in Your Experiments
What you want to measure Why it matters for your study Practical tip
Total β‑Catenin protein (by Western blot, IHC) Indicates whether the pathway is active at the protein level. Run a loading control (e.g., GAPDH). Compare with untreated vs treated samples.
Phosphorylated β‑Catenin (Ser33/37/T41) Reflects turnover; high levels suggest rapid degradation, low levels indicate stabilization. Use phospho‑specific antibody; ensure that the sample is freshly lysed and protease/phosphatase inhibitors are present.
Total mRNA of target genes (by qPCR or RNA‑seq) Confirms transcriptional activation downstream of the pathway. Normalize to housekeeping gene (e.g., ACTB).
Cellular localization (immunofluorescence, subcellular fractionation) Shows nuclear translocation of β‑catenin; a hallmark of active signaling. Include cytoplasmic and nuclear markers as controls.
3. Practical checklist for a typical WNT/β‑catenin assay
Step What to do Why it matters
1. Cell preparation Seed cells at ~70 % confluency; treat with DMSO or appropriate inhibitor (e.g., XAV939) for 24–48 h. Ensures cells are in a comparable state before assay.
2. Harvest Wash with PBS, add lysis buffer (RIPA + protease inhibitors). Prevents protein degradation and preserves phosphorylation status.
3. Protein quantification Use BCA or Bradford to measure total protein. Allows loading equal amounts on gel for comparison.
4. SDS‑PAGE & transfer Run 10–12 % gel, transfer to PVDF (semi‑dry or wet). Enables efficient antibody binding.
5. Blocking 5 % BSA in TBST for phospho‑antibody; 3 % milk for total protein. Reduces non‑specific binding.
Incubate overnight at 4 °C. | Allows detection of phosphorylated sites and normalization. | | 7. Secondary antibody | HRP‑conjugated anti‑IgG (species‑specific), 1:5000, 1 h RT. | For chemiluminescent signal. | | 8. Development | ECL substrate, expose to X‑ray film or CCD camera for 10–60 s depending on signal intensity. | Visualize bands corresponding to MAPK phosphorylation. |
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Notes
The above protocol can be adapted to other post‑translational modifications (e.g., acetylation, ubiquitination) by changing the primary antibody accordingly.
For mass‑spectrometry based PTM mapping, additional enrichment steps (e.g., phosphopeptide enrichment using TiO₂ or IMAC) would be required after cell lysis and protein digestion.
