They were then layered over a 1 ml cushion of lysis buffer containing 0.88 M sucrose and then collected by centrifugation at 5000×g for 10 minutes.
In other experiments, HK-2 cells were incubated with 0.1, 1 or 10 μM digitoxin for 24 h and then collected using RIPA buffer.
For immunoblot analysis, the cells were treated with the indicated agents and then collected in lysis buffer (Cell Signaling, Danvers, MA, USA).
After 24, 48 and 72 h of treatment, the cells were washed in cold PBS to slow biochemical reactions, and then collected in homogenising buffer (HB) (20 mM Tris, 1 mM DTT, 2 mM ATP and 5 mM MgCl2, pH 7.2), and centrifuged at 10,000 rpm for 15 min at 4°C.
MCF-7 cells were washed in ice cold PBS and then collected in lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, and 1% Triton X-100) containing protease inhibitors (Sigma-Aldrich) and RNase OUT (Invitrogen).
Cultured cells were starved in serum-free DMEM medium for 48 hours and then collected with lysis buffer (1 mM CaCl2, 1 mM MgCl2, 1% NP-40, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μM PMSF, and 100 μM NaVO4).
The cell monolayer was washed three times with PBS, and then collected into 1 mL homogenizing buffer (0.25 M sucrose in 10 mM Tris HCl pH 7.4).
Briefly, cells (1 × 10) were transfected with Pygo2 siRNA plasmids and scrambled control, and then collected and resuspended in binding buffer.
Catheters were then bisected longitudinally and biomass was removed from internal cavities and external bodies by forcefully scraping each site with a scalpel, rinsing the blade with 200 µl of TE/NP40 (10 mM Tris-pH 8.0/1 mM EDTA/0.1% NP40), and then collecting the buffer and biomass in separate, sterile microcentrifuge tubes.
