The cells were then treated with 1 μM ionomycin for 30 minutes, after which time they were either fixed immediately or washed extensively with PBS and incubated for 6 hours in medium lacking the drug ("washout").

Membranes were stripped at 50 C in, washed extensively with TBST, blocked and hybridized as described above using a mouse anti-actin primary antibody at 1:1000 and HRP-conjugated goat anti-mouse IgG secondary antiserum as described above.

Subsequently, slides were gently agitated in 0.2% SDS (no salt) overnight at 42 C, washed extensively with water, scanned to ensure that the dye was completely removed, restained with Vistra Green and rescanned.

Rib cages were transferred to phosphate buffered saline (PBS) and washed extensively.

The cells were briefly hemolyzed, washed extensively in DMEM containing 10% FBS, filtered through a 100 μM sterile nylon filter, counted by hemacytometer, and suspended in complete medium.

After extensively washing with the same buffer, the column was eluted with buffer 3 plus 0.15 M (NH 4 )2 SO 4.