After the addition of lysis buffer, the cells were scraped off and keep on ice for 30 min. Protein concentration of lysates were measured by Bio-Rad protein assay kit after centrifugation at 14000 rpm for 10 min. Lysates with 30 µg of protein were subjected to SDS-PAGE for CD44 detection.
To obtain SDS lysates cells were washed three times with PBS at room temperature, residual wash buffer was then removed and cells were scraped with boiling SDS-PAGE sample buffer, followed by 5 min boiling in a hot block.
For electron microscopy, cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4), then cells were scraped in PB and post-fixed with 1% osmium tetroxide in PB containing 0.8% potassium ferricyanide at 4°C.
The cells were then washed five times with PBS, MPER buffer (1.1 mL) was added, the cells were scraped, and the suspension was frozen in N2 and thawed twice.
Ice-cold nuclei EZ lysis buffer (2 ml) was added, and the cells were scraped with a small bladed cell scraper.
Cells were scraped in homogenization buffer (1 mM EDTA, 1 mM EGTA, 10 mM Tris-HCl, pH: 7.5, 1 µg/ml leupeptin, 100 µg/ml TPCK and 1 mM PMSF) and homogenized by using a Dounce homogenizer.
Caco-2/TC7 cells were scraped into lysis buffer (20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.15 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) containing protease and phosphatase inhibitor cocktails.
After the proper treatment, cells were resuspended in 50 ul of chilled lysis buffer on ice for 10 min, and cells were scraped off from the plates and centrifuged at 8,000 rpm for 1 min. The supernatnat was transferred to a microtube, and 50∼100 ug of protein was diluted to 50 ul cell lysis buffer for each assay.
Cells were scraped into RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.5 mM PMSF, complete protease inhibitor cocktail (Calbiochem), 50 mM NaF and 0.2 mM sodium orthovanadate) and sonicated to generate ∼500 nt chromatin fragments.
After rinses in this buffer with 1 % bovine serum albumin, cells were scraped off with a rubber policeman, and centrifuged with 3%% glutaraldehyde.
