Ab109429

After 48 h of transfection, cells were washed in cold PBS for three times and then lysed on ice with whole cell lysate for 10 min, with the concentration of the product sequentially determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 30 μg of the total proteins was separated by polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham, USA). After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK). PBST (PBS buffer containing 0.1% Tween-20) was used to wash the membranes after each reaction. An optical luminometer (GE, USA) was employed to visualize the protein bands.

All proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer and a protease inhibitor in a 100:1 ratio. Samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into PVDC membrane (Millipore, Massachusetts, USA) for 2h. Then, the membranes were incubated with primary antibody at 4 °C overnight. Tris-HCl solution + Tween-20 (TBST) was used to wash the membranes three times for 10 mins. Subsequently, they were incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature. Finally, the blots were detected with enhanced chemiluminescence, and band intensities were measured with Quantity-One software (Bio-Rad, Hercules, CA, USA). The primary antibodies against IFITM3 (ab109429), Bax (ab32503), Bcl2 (ab32124), tubulin (ab15246), GAPDH (ab8245), N-cadherin (ab76011), and E-cadherin (ab40772) were purchased from Abcam. Western blotting was used to calculate the amount of expressed protein.