2.5. Western Blotting

Cells were lysed with RIPA lysis buffer, and the total protein concentration in each supernatant was determined using a BCA Protein Assay Kit (Pierce Biotechnology, Waltham, MA, USA). Next, a 20 μg sample of total protein from each supernatant was loaded onto a 12% SDS-PAGE gel and separated at 80 V for 40 minutes. The protein bands were then transferred onto PVDF membranes, which were subsequently blocked with 10% nonfat milk. Next, the membranes were incubated with anti-TSG101 antibodies (Cat#: BM4821, 1 : 1000, Boster, China), anti-CD63 antibodies (Cat#: PB9250,1 : 1000, Boster, China), anti-GRP94 antibodies (Cat#: PROTP14625, 1 : 1000, Boster, China), anti-ROBO1 antibodies (Cat#: A01530-2, 1 : 1000, Boster, China), anti-SRGAP2 antibodies (Cat#PA5-55792, 1 : 1000, Invitrogen, USA), and anti-GAPDH antibodies (Cat#A00227-1, 1 : 1000, Boster, China) at 4°C overnight. Next, the membranes were incubated with secondary antibodies at room temperature for an additional 1 h. The immunostained protein bands were visualized with Pierce ECL Western Blot Substrate (Merck, USA).