Ab229832

Cells were lysed in an ice bath for 30 min in NP40 lysis buffer (50 mmol/L Tris-HCI pH 7.4, 150 mmol/L NaCl, 1% NP-40, 1 mmol/L EDTA) containing protease and phosphatase inhibitors (5 mmol/L PMSF, 3 mmol/L NaF, 1 mmol/L DTT, 1 mmol/L NaVO₄). Proteins in 20 μg of protein cell lysate were separated by SDS-PAGE and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membrane was then washed in PBS, blocked for 1 h (5% milk-TBS-0.05% Tween 20), and incubated with primary antibody overnight at 4 °C. The following primary antibodies were used: anti-GAPDH antibody (Abcam.ab181602, 1:1000), anti-ETV7 antibody (Abcam.ab229832, 1:1000), anti-FOXO3 antibody (Abcam.ab47285, 1:1000), anti-FOXO4 antibody (Abcam.ab128908, 1:1000), and anti-c-MAF antibody (Abcam.ab77071, 1:1000). The membrane was washed with PBS and incubated with an appropriate amount of secondary antibody for 1 h at room temperature (Li-Cor, IRDye 600LT; IRDye 800CW, 1:10000). The membrane was visually analyzed using an imaging system (ChemiDocXRS+, BIO-RAD). Uvitec Alliance software (Eppendorf, Hamburg, Germany) was used to quantify the data. All data were from three independent biological repeats.