Ab221939

Cell lysates were resuspended with reaction buffer (5 mmol/L ATP, 1 mmol/L spermine, 25 mmol/L KCl, 5 mmol/L MgCl₂, 1 μmol/L ³H proline, 50 mmol/L HEPES‒KOH [pH 7.6], glutamate or leucine [60 Ci/mmol, Perkin‐Elmer]), and incubated with GOT1 (ab221939) or MDH1 (ab180152, Abcam Inc.) antibody. Aliquots (15 μL) of immunoprecipitated proteins were quenched on Whatman filter paper, while radioactivity was quantified using Beckman Coulter LS6500 (Beckman Conlter, Inc.).

Immunohistochemistry was performed on rat brain microvascular endothelial cells treated with 10% serum exosome collected from control, model, and model + miR-9-5p angomir rats. The paraffin-embedded section of the cerebral cortex was fixed in 4% paraformaldehyde for 24 h and washed with phosphate-buffered saline (PBS). Subsequently, the sections were immersed in 0.5% triton for 20 min, washed with PBS, incubated with 10% goat serum for 30 min, and incubated overnight at 4 °C with primary antibodies of TFRC (ab214039; Abcam, Cambridge, MA, USA) and GOT1 (ab221939; Abcam). Then, the sections were washed by PBS and incubated with the secondary antibody at 37 °C for 30 min. Again, the sections were washed with PBS and deparaffinized using anti-fluorescence quenching mounting tablets containing 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), kept at 4 °C in dark, and watched on a confocal laser microscope (Leica).