After 1 hr, insoluble material was pelleted by centrifugation at 180,000× g for 40 min. The clarified supernatant was incubated with NiNTA resin for 1 hr and washed thoroughly with membrane buffer supplemented with 4 mM β-mercaptoethanol (BME), 20 mM imidazole, and 1% Triton X-100.
After specific treatment, oocytes were washed thoroughly and cultured in M16 supplemented with 10% fetal bovine serum (FBS) (Gibco) to (MI (8 hours) or MII (14 hours).
After specific treatment, oocytes were washed thoroughly and cultured in M16 supplemented with 10% fetal bovine serum (FBS) (Gibco) to GV (0 hour), GVBD (2 hours), Pro-MI (5 hours), MI (8 hours), ATI (9.5 hours), and MII (12 14 hours) stages.
Thin pyramids were cut from the kidney and incubated in 0.006% liberase solution for 20-25 min at 30°C and thoroughly rinsed in microdissection solution supplemented with a cocktail of protease inhibitors (Complete EDTA-free, Roche Diagnostics) when used for western blot.
The sections were thoroughly rinsed in PBS-T (PBS supplemented with 0.1% Tween-20) in between the above mentioned steps.
After the overnight incubation, cells were thoroughly washed and cultured in complete DMEM medium supplemented with sodium butyrate for 8 hours.
The fatty acids and BSA solutions were then removed, cell monolayers thoroughly rinsed with PBS and incubated with fresh αMEM supplemented with 2% FBS for another 24 h.
In this case, β-cyclodextrin (4.0 mM) was supplemented as a cell membrane transfer agent; the culture was thoroughly shaken and then divided into 1.0 mL aliquots.
Internal solution was supplemented with 0.6 mM GTP and 2 mM ATP and mixed thoroughly just prior to use.
The binding buffer was supplemented with 0.1 M NaCl to prevent non-specific interactions, and the resin was thoroughly washed before the elution.
