Trans blotr turbo transfer system

Western blotting according to standard procedure. The difference is that the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Trans-BlotR TurboTM Transfer SYSTEM, BIO-RAD, USA) was then used to transfer total protein onto polyvinylidene fluoride membranes (Roche, USA). The following primary antibodies were used: anti-EMP3 (Abcam), α-tubulin, anti-MMP2, anti-MMP9, and anti-N-cadherin from Proteintech Group, Inc. ImageJ was used to perform densitometric analyses of immunoblots.

Total proteins were isolated from microglia cultures using Pierce^TM RIPA buffer (Thermo Fisher Scientific, Dreieich, Germany), and protein concentrations were measured using a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Dreieich, Germany) according to the manufacturer’s instructions. Lysates (10 µg total protein/lane) were loaded on Mini-PROTEAN Precast gels (Bio-Rad, München, Germany) for electrophoresis. Blotting was performed using a Trans-Blot^R Turbo^TM Transfer System and a Trans-Blot^R Turbo^TM RTA Midi PVDF Transfer Kit (Bio-Rad, München, Germany). All membranes were blocked with 5% BSA (Sigma-Aldrich) in TBST for 90 min. Incubation with primary antibodies against CD74 (BD Biosciences, 555317) and β-Actin (Cell Signaling Technologies, 4970) was performed at 4 °C overnight. Finally, membranes were washed with TBST and incubated with HRP-conjugated anti-rat (Abcam, ab97057) and anti-rabbit (Cell Signaling Technologies, 7074S) secondary antibodies. Labelled proteins were detected using a Pierce^TM ECL Western Blotting Substrate (Thermo Fisher Scientific, Dreieich, Germany). Blots were captured using a Proxima ECL Detection Setup (Isogen). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).