Bca protein concentration measurement kit

Protein lysates of Vector‐TEVs, Foe‐TEVs or Foe‐Th cells were prepared according to standard procedures, and the contents of proteins were assessed by the BCA protein concentration measurement kit (Thermo Fisher Scientific). Subsequently, the samples were isolated using Bis‐Tris gel (Invitrogen), and then transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked in TBS‐T in 5% BSA for 1 h, incubated with primary antibodies overnight at 4 °C, then rinsing the membranes with PBS. Then, the membranes were covered with secondary antibody. Incubated at room temperature for 2 h, signals were assessed by enhanced Chemiluminescence Advanced System (GE Healthcare). The primary antibodies involving against CD9 (1:1000, Abcam), CD63 (1:1000, Abcam), Tubulin (1:1000, Abcam), TSG101(1:1000, Abcam), CTLA‐4 (1:1000, Abcam), TGF‐β (1:1000, Abcam), IL‐10 (1:1000, Abcam), PDCD‐1 (1:1000, Abcam), CD35 (1:1000, Abcam), CD73 (1:1000, Abcam), LAG3 (1:1000, Abcam), TIGIT (1:1000, Abcam), calnexin (1:1000, Abcam), histone 3 (1:1000, Abcam), and GM13 (1:1000, Abcam), ITGA4 (1:800, Abcam), ITGAL (1:800, Abcam), ITGB1 (1:800, Abcam), ITGB2 (1:800, Abcam) and secondary antibody Alexa Fluor Plus 800 (1:10 000, Thermos) were used.

Total protein was extracted from cell lysates with a protease inhibitor. A BCA protein concentration measurement kit (Thermo Fisher Scientific, Shanghai, China) was used to determine protein concentration. Proteins were separated using 4–20% SDS-PAGE, and then transferred to a PVDF membrane, which was then blocked with 5% BSA for 1 h at room temperature followed by incubation overnight at 4 °C with the primary antibodies E2F2 (1:1000, Santa Cruz Biotechnology) and E2F7 (1:1000, Proteintech). Following six washes with TBST, the membrane was incubated with a second antibody (Abways, Shanghai, China) for 1 h, and relative protein expression was determined by electrochemiluminescence.