Gel transfer device

Cells were dissociated and then pelleted by centrifugation at 1000 rpm/min for 5 min. The supernatant was removed, and the cell was lysed using Beyotime (# P0013B) lysis buffer. The lysate was vigorously shaken for 15 s and then placed on ice for 10 min. This step was repeated 3 times. After determining the protein concentration using the BCA method, 5X loading buffer was added, and the protein was denatured at 100 °C for 5 min. Electrophoresis was performed using 10% SDS-PAGE gels, and the samples were transferred to PVDF membranes at 300 mA for 90 min using a gel transfer device (Bio-Rad, USA). After blocking the PVDF membrane with 5% non-fat milk for 1 h at RT, the membrane was washed three times with TBST and then incubated with primary antibodies overnight at 4℃. The following day, the membranes were washed three times with TBST and then incubated with secondary antibodies, including Goat anti-Rabbit IgG(H + L) IRDye 800CW or Goat anti-mouse IgG(H + L) IRDye 800CW(LI-COR, USA) for 1 h at 37℃. Finally, the images were acquired using a UVA Bio imaging System (LI-COR Biosciences, USA).

Cells were rinsed in PBS (Corning, USA) and harvested in a CardioEasy CM dissociation buffer (Cellapy). The cells were then rinsed again in PBS and pelleted by centrifugation twice at 1200 rpm for 5 min each time. After removing the supernatant, SDS-PAGE protein-loading buffer (Beyotime, China) was added, and the cells were lysed by sonication and heat-denatured. According to the molecular weight of the protein, we configured a 10% separation gel and a 4% concentration gel for electrophoresis and performed gel transfer to a polyvinylidene difluoride membrane using a gel transfer device (Bio-Rad) over 120 min. Then, we blocked the membrane with 5% skimmed milk for 1 h at room temperature. The membrane was incubated with the primary antibody overnight at 4 °C, followed by incubation with the secondary antibody for 2 h at room temperature.