Materials and Reagents

1. 10 cm Petri dishes
   
2. 50 ml Beckel centrifuge tubes
   
3. 1.5 ml microcentrifuge tubes
   
4. Amicon Ultra-15 centrifugal filter
   
5. 0.45 µm immobilon-FL PVDF (EMD Millipore, catalog number: IPFL00010 )
   
6. Wild type MEF and Rab8a^-/- MEF cell lines
   
7. MEF isolation and phenotypic analysis was previously described (Das et al., 2015)
   
8. DEME with glucose, L-glutamine and sodium pyruvate (Mediatech, catalog number: 10-013 )
   
9. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442-500ML )
   
10. Pen-strep (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
    
11. Lactalbumin hydrolysate solution (50x) (Sigma-Aldrich, catalog number: 58901C-100ML )
    
12. NaN₃
    
13. NaF
    
14. Na₃VO₄
    
15. PMSF
    
16. DTT
    
17. Nonidet^® P40 substitute (Sigma-Aldrich, catalog number: 74385-1L )
    
18. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
    
19. Tween^® 20 (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP337-500 )
    
20. Sodium chloride (NaCl)
    
21. Potassium chloride (KCl)
    
22. Disodium hydrogen phosphate (Na₂HPO₄)
    
23. Potassium dihydrogen phosphate (KH₂PO₄)
    
24. Protease inhibitor cocktail tablets (Sigma Aldrich, catalog number: 11873580001 )
    
25. Skim milk powder (EMD Millipore, catalog number: 1.15363.0500 )
    
26. Amersham ECL Western blotting detection reagents (GE Healthcare, catalog number: RPN2209 )
    
27. Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) (GE Healthcare, catalog number: NA934V-1ML )
    
28. Wnt5a/b (C27E8) Rabbit mAB (Cell Signaling Technology, catalog number: 2530S )
    
29. Histone H3 (D1H2) XP^® Rabbit mAb (Cell Signaling Technology, catalog number: 4499S )
    
30. 10% FBS DMEM (see Recipes)
    
31. 1x LAH DMEM (see Recipes)
    
32. Non-denaturing lysis buffer (see Recipes)
    
33. 1% Tween 20 in PBS (PBST) (see Recipes)
    
34. 5% skim milk in PBST (see Recipes)
    
35. 1x running buffer (see Recipes)
    
36. 1x transfer buffer (see Recipes)
    

Equipment

1. Thermo IEC Centra CL3R Bench-model, Refrigerated centrifuge
   
2. Avanti J-26 XP centrifuge (Beckman Coulter, model: Avanti J-26XP )
   
3. Beckman JA-25.50 rotor (Beckman Coulter, model: JA-25.50 )
   
4. Bio-Rad Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories, model: 1658005EDU )
   
5. XCell SureLock^® Mini-Cell and XCell II blot module (Thermo Fisher Scientific, Novex^TM, catalog number: EI0002 )
   
6. QSonica XL-2000 (Qsonica, model: XL-2000 )
   
7. Ultrospec 2100^® UV-Visible spectrophotometer (Biochrom, model: 80-2112-21 )
   

Procedure