Total protein was extracted using RIPA lysis buffer containing a protease
inhibitor mixture. The protein samples were separated by SDS-PAGE and
transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were
blocked for 1 hour with 5% bovine serum albumin and incubated with primary
antibody overnight at 4°C. The following day, the membranes were washed with
Tris-buffered saline containing Tween-20, followed by incubation with a
secondary antibody (1:1000 dilution for both anti-mouse and anti-rabbit
antibodies) for 1 hour. Protein bands were visualized using a chemiluminescence
detection system, with vinculin serving as a loading control.
As primary antibodies, we used anti-FUT5 (1:1000 dilution) (Biorbyt, orb449427),
anti-vinculin (1:1000 dilution) (ABclonal, A2752), anti-ITGB3 (1:3000 dilution)
(Abcam, ab197662), anti-Ki-67 (1:1000 dilution) (Abcam, ab16667),
anti-E-cadherin (1:20 000 dilution) (Proteintech, 20874-1-1AP), and anti-GAPDH
(1:2000 dilution) (Abcam, ab8245). Goat anti-rabbit IgG and goat anti-mouse IgG
secondary antibodies were purchased from Beijing Solarbio Science and Technology
Co., Ltd.
inhibitor mixture. The protein samples were separated by SDS-PAGE and
transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were
blocked for 1 hour with 5% bovine serum albumin and incubated with primary
antibody overnight at 4°C. The following day, the membranes were washed with
Tris-buffered saline containing Tween-20, followed by incubation with a
secondary antibody (1:1000 dilution for both anti-mouse and anti-rabbit
antibodies) for 1 hour. Protein bands were visualized using a chemiluminescence
detection system, with vinculin serving as a loading control.
As primary antibodies, we used anti-FUT5 (1:1000 dilution) (Biorbyt, orb449427),
anti-vinculin (1:1000 dilution) (ABclonal, A2752), anti-ITGB3 (1:3000 dilution)
(Abcam, ab197662), anti-Ki-67 (1:1000 dilution) (Abcam, ab16667),
anti-E-cadherin (1:20 000 dilution) (Proteintech, 20874-1-1AP), and anti-GAPDH
(1:2000 dilution) (Abcam, ab8245). Goat anti-rabbit IgG and goat anti-mouse IgG
secondary antibodies were purchased from Beijing Solarbio Science and Technology
Co., Ltd.