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docs/Experiments/EXP-0224-YBX1-CEBPA-CoIP-3T3-adipogenesis.md

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experiment_id, title, date, researchers, protocol_id, protocol_name, status, aim, project
experiment_id title date researchers protocol_id protocol_name status aim project
EXP-0224 YBX1-CEBPA Protein Interaction in Early Adipogenesis (Iteration 1) 2025-04-11
james-m-jordan
linda-onsei
PROT-0036 Adipogenic Induction Treatment completed Investigate physical interaction between YBX1 and C/EBPα during early adipogenesis (24h post-induction) in 3T3 cells using reciprocal co-immunoprecipitation Transcriptional Regulation in Early Adipogenesis

SAMPLE METADATA

cell_lines:

  • name: "3T3" media: "DMEM high glucose + 10% FBS + 1% Pen-Strep" passage: "P8-P12" plate_format: "25 cm flasks" condition_map: | Dish 1-3: 3T3 + Control medium (24h) Dish 4-6: 3T3 + Adipogenic induction medium (24h) replicates: 2 sample_location: "-80°C freezer, rack 1-1-4-A"

REAGENTS & INSTRUMENT SETTINGS

adipogenic_induction: reagents: - name: "IBMX" concentration: "0.5 mM" supplier: "Sigma-Aldrich (I5879)" - name: "Dexamethasone" concentration: "1 µM" supplier: "Sigma-Aldrich (D4902)" - name: "Insulin" concentration: "10 µg/mL" supplier: "Sigma-Aldrich (I6634)" cell_lysis: buffer: "RIPA Buffer with protease inhibitors" volume: "500 µL per dish" incubation: "30 min on ice with occasional vortexing" co_immunoprecipitation: antibodies: - name: "Anti-YBX1" amount: "2 µL of 1:1000 dilution per IP" supplier: "Abcam (ab268094)" - name: "Anti-C/EBPα" amount: "2 µL of 1:1000 dilution per IP" supplier: "Abcam (AB317442)" - name: "Normal Mouse IgG (control)" amount: "2 µL of 1:1000 dilution per IP" supplier: "Cell Signaling Technology (#5415)" beads: "Protein A/G beads (NEB S1430 and S1425 mixed 1:1)" volume: "30 µL per IP" binding: "Overnight at 4°C with rotation" ip_wash_buffer: "PBS + 0.02% Tween-20" elution_buffer: "Non-denaturing elution buffer, 20 µL per sample" western_blot: gel: "Invitrogen NuPAGE 4-12% Bis-Tris mini gels (15-well)" transfer: "iBlot 3 Dry Blotting System (P0 program, 7 min)" antibody_detection: "iBind 3 Western System" ibind_protocol: "2 µL of 1:1000 primary antibody dilution per 2 mL iBind solution per mini gel" primary_antibodies: - name: "Anti-YBX1" dilution: "1:1000" supplier: "Abcam (ab268094)" - name: "Anti-C/EBPα" dilution: "1:1000" supplier: "Abcam (AB317442)" secondary_antibody: "Anti-mouse HRP, 1:5000" imaging: "ChemiDoc Imaging System" instruments:

  • name: "Invitrogen iBlot 3" settings: "P0 program, 7 minutes"
  • name: "Invitrogen iBind 3" settings: "Standard protocol, 3 hours"
  • name: "ChemiDoc Imaging System" settings: "Chemiluminescence, auto-exposure"

1️⃣ Experiment Timeline & Execution

Day 1: 2025-04-11

  • Seed 3T3 cells in six 10 cm dishes at density of 5 × 10⁵ cells/dish
  • Incubate overnight at 37°C, 5% CO₂
  • Prepare stock solutions for adipogenic induction medium

Day 2: 2025-04-12

  • Verify cells are ~90% confluent
  • Prepare fresh adipogenic induction medium:
    • IBMX (0.5 mM)
    • Dexamethasone (1 µM)
    • Insulin (10 µg/mL)
  • Replace media:
    • Dishes 1-3: Regular complete medium (control)
    • Dishes 4-6: Adipogenic induction medium
  • Incubate for 24 hours at 37°C, 5% CO₂

Day 3: 2025-04-13

  • Harvest cells from all dishes:
    • Wash twice with ice-cold PBS
    • Add 500 µL RIPA buffer with protease inhibitors per dish
    • Scrape cells and collect lysate
    • Incubate 30 min on ice with occasional vortexing
    • Centrifuge at 14,000 × g for 15 min at 4°C
    • Transfer supernatant to new tubes

BCA Protein Assay Procedure

  • Prepare BSA standards (0, 0.125, 0.25, 0.5, 1, 2 mg/mL) in the same buffer as samples
  • Dilute samples 1:5 in RIPA buffer (5 µL sample + 20 µL buffer)
    • CRITICAL INPUT: Researcher must record total lysate volume before dilution for each sample!
    • CRITICAL INPUT: Researcher must record dilution factor used for BCA assay!
  • Add 200 µL BCA working reagent (50:1, Reagent A:B) to 10 µL of each standard/sample in a 96-well plate
  • Incubate plate at 37°C for 30 minutes
  • Measure absorbance at 562 nm
  • Generate standard curve by plotting absorbance vs. known BSA concentrations
  • Calculate protein concentration of each sample using the standard curve equation
  • Adjust for dilution factor: Final concentration = Measured concentration × Dilution factor

Sample Preparation for Co-Immunoprecipitation

  • Prepare samples for co-immunoprecipitation:
    • 500 µg protein per IP reaction
    • 2 samples per condition (control and adipogenesis induction)
    • 2 IPs per sample (YBX1 pull-down and CEBPA pull-down)
  • Preclear lysates with 50% beads:
    • Add 20 µL of 50% Protein A/G beads (NEB S1430 and S1425 mixed 1:1) to each lysate
    • Incubate at 4°C with rotation for 1 hour
    • Centrifuge at 2,500 × g for 5 minutes or place on magnetic stand
    • Transfer precleared supernatant to new tubes
  • Add antibodies to precleared lysates (2 µL of 1:1000 dilution each):
    • Anti-YBX1
    • Anti-C/EBPα
    • Normal Mouse IgG (control)
  • Incubate overnight at 4°C with rotation

Day 4: 2025-04-14

  • Add 30 µL Protein A/G beads (NEB S1430 and S1425 mixed 1:1) to each IP sample
  • Incubate overnight at 4°C with thermomixing at 400 rpm
  • Wash beads 4× with IP wash buffer (PBS + 0.02% Tween-20):
    • Add 500 µL wash buffer to each sample
    • Invert tubes several times or mix gently
    • Place on magnetic stand to separate beads
    • Remove supernatant carefully without disturbing beads
    • Repeat 3 more times (4 washes total)
  • Elute proteins with non-denaturing elution:
    • Add 20 µL non-denaturing elution buffer to each sample
    • Incubate at 37°C with rotation for 5 minutes
    • Place tubes on magnetic stand to separate beads
    • IMPORTANT: Carefully collect supernatant without disturbing beads
    • WARNING: DO NOT load any beads into the gel - they will interfere with migration!
  • Load samples on Invitrogen NuPAGE 4-12% Bis-Tris mini gels (2 gels):
    • Input (10% of lysate)
    • IP samples
    • Gel 1: YBX1 pull-down probed with anti-CEBPA
    • Gel 2: CEBPA pull-down probed with anti-YBX1
  • Run gels at 150V for 1 hour
  • Transfer to PVDF membranes using iBlot 3 (P0 program, 7 min)
  • Process membranes on iBind 3:
    • Prepare iBind solution: 2 µL of 1:1000 primary antibody dilution per 2 mL iBind solution per mini gel
    • Gel 1: Probe with anti-CEBPA antibody
    • Gel 2: Probe with anti-YBX1 antibody
  • Image blots on ChemiDoc system
  • Quantify band intensity using ImageJ

Gel Loading Instructions (15-well gels)

Gel 1: YBX1 Pull-Down (Probed with anti-CEBPA)

Loading order for 15-well gel:

  1. Protein ladder
  2. IgG PD - Control condition (no adipogenic induction)
  3. IgG PD - Adipogenic induction
  4. YBX1 PD - Control condition (no adipogenic induction)
  5. YBX1 PD - Adipogenic induction
  6. Protein ladder
  7. Input - Control condition (no adipogenic induction)
  8. Input - Adipogenic induction
  9. Protein ladder
  10. Leftover - IgG PD Control
  11. Leftover - IgG PD Adipogenic
  12. Leftover - YBX1 PD Control
  13. Leftover - YBX1 PD Adipogenic
  14. Empty
  15. Empty

Gel 2: CEBPA Pull-Down (Probed with anti-YBX1)

Loading order for 15-well gel:

  1. Protein ladder
  2. IgG PD - Control condition (no adipogenic induction)
  3. IgG PD - Adipogenic induction
  4. CEBPA PD - Control condition (no adipogenic induction)
  5. CEBPA PD - Adipogenic induction
  6. Protein ladder
  7. Input - Control condition (no adipogenic induction)
  8. Input - Adipogenic induction
  9. Protein ladder
  10. Leftover - IgG PD Control
  11. Leftover - IgG PD Adipogenic
  12. Leftover - CEBPA PD Control
  13. Leftover - CEBPA PD Adipogenic
  14. Empty
  15. Empty

2️⃣ Raw Data & Resources

IMPORTANT: All raw data files MUST be placed in the directory Data/EXP-0224/raw/ and listed in the table below!

Filename Description Date Added
BCA_protein_assay.xlsx Protein concentration measurements and standard curve 2025-04-13
YBX1_pulldown_blots.tif YBX1 IP probed with anti-CEBPA (includes input samples) 2025-04-14
CEBPA_pulldown_blots.tif CEBPA IP probed with anti-YBX1 (includes input samples) 2025-04-14

3️⃣ Results & Analysis

Protein Concentration

Protein concentrations in cell lysates:

  • Control samples: 1.8-2.2 mg/mL
  • Adipogenic induction samples: 1.7-2.0 mg/mL

Co-IP Efficiency

The immunoprecipitation successfully pulled down the target proteins in all samples. YBX1 IP efficiency: ~80% depletion from lysate CEBPA IP efficiency: ~70% depletion from lysate

YBX1-CEBPA Interaction Analysis

The following table summarizes the quantified interaction data:

Sample YBX1 pulldown CEBPA pulldown IgG control
Control 0.15 ± 0.03 0.11 ± 0.02 0.02 ± 0.01
Adipogenic 0.94 ± 0.12 0.72 ± 0.09 0.03 ± 0.01
Fold change 6.3× 6.5× 1.5×

Analysis Notes

Analysis script: Analysis/EXP-0224_CoIP_quantification.R

  • Band intensity quantified using ImageJ
  • Interaction strength calculated as ratio of co-IPed protein to pulled-down protein
  • Statistical analysis: paired t-test between conditions (p < 0.001)

4️⃣ Interpretation

Summary of Findings

We observed a weak interaction between YBX1 and C/EBPα in control conditions that was significantly enhanced (>6-fold) after 24 hours of adipogenic stimulation. The reciprocal Co-IP (YBX1 pull-down and CEBPA pull-down) showed consistent results, providing strong evidence for the physical interaction of these proteins during the early stages of adipogenesis.

Relation to Project Goals

This experiment supports our hypothesis that YBX1 and C/EBPα physically interact during early adipogenesis. The dramatic increase in interaction after adipogenic stimulation suggests that this interaction may play an important role in initiating the transcriptional changes required for adipocyte differentiation.

5️⃣ Next Steps ✅

Check boxes when complete. These can auto-update TASKS.md.

  • Repeat Co-IP in a second iteration to validate findings (see EXP-0226)
  • Perform reciprocal Co-IP at multiple timepoints (6h, 12h, 24h, 48h)
  • Characterize binding domains through truncation mutants
  • Present results at lab meeting on 2025-04-18
  • Consider ChIP-seq to identify co-regulated genes

6️⃣ Team Discussion

Use this section for team comments, suggestions, and feedback.

james-m-jordan (2025-04-15): The interaction we observed is much stronger than expected based on the literature. Let's repeat this experiment to confirm our findings and potentially include more controls in the next iteration (EXP-0226).

linda-onsei (2025-04-15): I agree with James. For the next iteration, we should also check protein levels by straight Western blot to determine if the increased interaction is partly due to increased expression of either protein during adipogenesis.

james-m-jordan (2025-04-16): Excellent suggestion, Linda. We'll incorporate that into the design of EXP-0226. Also, I'm wondering if we should check for interactions with additional C/EBP family members in future experiments.

7️⃣ References & Related Experiments

  • Related protocol: Adipogenic Induction Treatment
  • Follow-up experiment: EXP-0226
  • Literature: Girard J, et al. (2018) YBX1 interacts with C/EBP transcription factors to regulate adipogenesis. Cell Reports 25:788-801.