Sc 100830

The total protein was extracted in the extraction buffer [20% glycerol, 50 mmol/L Tris-HCl (pH 6.8) and 0.5% (v/v) Tween 20], to which a protease inhibitor cocktail (Sigma-Aldrich, US) was added. Next, the samples were centrifuged at 12,000 rpm (6439 g) for 10 min to remove nondissolved material. The protein concentration in the supernatant was measured using a Tiangen protein assay kit (Tiangen Biotech, China). Aliquots of protein extract containing 30 µg of total protein were electrophoresed in a 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to a P-membrane (Millipore, US) for 3 h with transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, and 20% (v/v) methanol). The membrane was blocked with 2% dried nonfat milk and incubated with antibodies against L19 (sc-100830, Santa Cruz, US, 1∶500) and Stra8 (ab49602 Abcam, US, 1∶500). After treatment with the primary antibody, the membrane was washed in Tris-buffered saline-Tween buffer (20 mmol/L Tris, 500 mmol/L NaCl, 0.05% Tween 20) and incubated with the secondary antibody (dilution at 1∶3000). Final exposure was achieved using enzymatic chemiluminescence (Amersham Pharmacia, UK). The films were scanned and quantified using ImageJ software (n = 6/group).