Tanon 2500

The proteins in this study were lysed using RIPA Lysis Buffer (C05-01001, Bioss, China), followed by quantified with BCA Kit (A53225, ThermoFisher, USA). 20 μg of protein was taken to perform electrophoresis with SDS-PAGE, and then transferred to PVDF membrane (IPVH00010, Millipore, USA). After sealing with blocking Buffer (37565, Thermo Scientific) for 2 h, membranes were then incubated with primary antibodies (4°C, overnight). Afterwards, membranes were incubated with secondary antibodies. Protein bands were visualized with ECL luminous fluid (WBKlS0100, Millipore) and analyzed with gel imaging system (Tanon 2500, Solarbio, China) and Quantity One image analysis software (Bio-Rad, USA). All antibodies produced by Abcam (UK) were as follows: E-Cadherin (ab40772, 1/10000, 97 kDa); N-Cadherin (ab18203, 1/10000, 130 kDa); Vimentin (ab92547, 1/1000, 54 kDa); E2F7 (24489-1-AP, 1/1000, 90 kDa, Proteintech, USA); GAPDH (ab8245, 1/5000, 36 kDa); Goat Anti-Rabbit (ab205718); Goat Anti-Mouse (ab205719).

The proteins of BV2 microglia or mouse tissues were lysed on ice for 10 min using RIPA lysate (P0013, Beyotime, China), followed by quantification using the BCA method (A53227, ThermoFisher, USA). 20 μg of the protein was subjected to electrophoresis by SDS-PAGE, and then transferred to PVDF membrane (YA1701, Solarbio, China). After being sealed for 1 h, the membrane was incubated with primary antibodies (4 °C, overnight), and then incubated with corresponding secondary antibodies (room temperature, 2 h). Protein bands were visualized with ECL detection reagent (SL1350-100ml, Coolaber, China) and analyzed with gel imaging system (Tanon 2500, Solarbio, China) and Quantity One image analysis software (Bio-Rad, USA). The antibodies used were as follows: IL-1β (ab234437, Abcam, 1/1000, 30 kDa); TNF-α (ab255275, 25 kDa, 1/1000); caspase-3 (1/500, ab32351, 35 kDa); β-actin (ab8226, 1 µg/mL, 42 kDa); HMGB1 (ab79823, 1/10,000, 25 kDa); Goat Anti-Rabbit (ab205718); Goat Anti-Mouse (ab205719).