Anti rat

For anti-GFAP and anti-CD45 immunohistochemistry, brain sections were permeabilized with citrate buffer (pH 6) and with TBS-0.5% Triton-0.1% sodium azide. Blocking was performed in TBS-0.2% Triton-0.1% Sodium azide-10% goat serum for 1 h. Then, the tissues were incubated with rabbit anti-GFAP (840001, 1/2000, Biolegend, San Diego, CA, USA) or rat anti-CD45 (103102, 1/200, Biolegend) antibodies overnight in TBS-0.2% Triton-0.1% sodium azide-1% goat serum. The next day, the tissues were incubated with their respective anti-rabbit (Vector Laboratories Inc., Newark, CA, USA) or anti-rat (Biolegend) secondary antibodies and stained with DAPI. Brain sections were mounted on slides using Vectashield (Vector Laboratories Inc., Newark, CA). Negative controls were incubated without the primary antibody. Tissues were analyzed by fluorescent microscopy.

HEK293 cells were transfected with a FLAG-N expression plasmid by using Lipofectamine 2000 (Invitrogen). At 24 hpi, cells were harvested by trypsinization and centrifugation. After two washes with PBS, cells were swollen with Hypotonic Lysis Buffer (20 mM HEPES pH 7.4, 10 mM NaCl, 3 mM MgCl₂, and 1× Protease inhibitor (Gold Biotechnology)) and lysed with 27G needle in presence of 25 μg/mL digitonin. Digitonin is a mild detergent that has been shown not to lyse nuclei [38 (link)]. Insoluble, nuclear fraction was pelleted by centrifugation for 10 min at 1500× g, washed with PBS, and lysed with NuPAGE LDS Sample Buffer (Invitrogen). Soluble, cytoplasmic fraction was spun down for 15 min at 10,000× g, and the clear supernatant was kept for analysis. Samples were electrophoresed under reducing and denaturing conditions, and transferred onto a PVDF membrane overnight. Antibodies used for the blotting were anti-FLAG (Sigma, F3165, 1:2000), anti-GAPDH (BioLegend, 607901, 1:1000), anti-histone H2B (BioLegend, 606301, 1:1000), anti-mouse (Sigma, A2554, 1:6000), and anti-rat (BioLegend, 405405,1:2000).