Anti-NFAT4/NF-ATc3 (phospho S165)抗体(ab59204)

The cell treatment protocol is as follows:

a. HeLa cells were plated on flasks or dishes containing growth medium (basic medium containing 10% serum).
b. When the cells were at the log phase and 80-90% confluent, the culture media was drained. Wash cells with fresh basic media (no serum) once.
c. Add fresh basic media to the vessels and starve the cells for 18-24 hours.
d. Drain the cultured medium, then add fresh basic medium (about 4ml per T75 flask) to the vessels.
e. Add Ca2+ to the medium at 40nM.

f. Cells were incubated at 37°C for 30 mins in a CO2 incubator and then were used to prepare cell lysates.



Cell lysates were prepared as follows:
a. For adherent cells, wash cells with Wash Buffer (4℃) once. Add 5ml Wash Buffer (4℃) per T75 flask and immediately scrape the cells off with a rubber scraper. Collect cells by centrifugation (3000rpm, 5min) at 4℃ and remove Wash Buffer.
b. Leave the tube containing cells on ice for 10 minutes. Then add Cold (4℃) Lysis Buffer to the cells (150ul per 1X106-1X107cells).
c. Mix the cells in the buffer and roll tube continuous on ice for 30 minutes at 4℃.
d. (Optional) Sonication for 4X8 at output level 5 for whole cell lysis.
e. Collect the supernatant by centrifugation (>12000rhm, 10minutes) at 4℃. Assay protein concentration by BCA. The cell lysis can be stored at -70℃ or ready to use.

Wash buffer formula: 10 mM PBS with 1 mM NaF and 1 mM Na3VO4. Lysis buffer formula: 50 mM Kcl with 1% NP40, 25 mM HEPES, 125 uM DTT, and protease/phosphatase inhibitors (100 ug/mL leupeptin, 20 ug/mL aprotinin, PMSF 1 mM, Na3VO4 1 mM).