Anti nf κb p65 acetyl k310

Brain microvessels were smeared on slides, fixed for 10 min at 98 °C and then for additional 10 min at room temperature in 4 % paraformaldehyde. After washing 3 times with chilled PBS, slides containing isolated microvessels were permeablized with 0.1 % Triton X-100 (Sigma) for 30 min at room temperature, then rinsed with PBS. Slides were blocked using 3 % bovine serum albumin (BSA) in PBS for 1 h at room temperature followed by overnight incubation at 4 °C with anti-NF-κB p65 (acetyl K310, Abcam, Cambridge, MA, USA) at 1:200 dilution. Following washes with PBS, α-rabbit Alexa Fluor 488 (Invitrogen, Camarillo, CA, USA) was applied at 1:400 dilution in 3 % BSA in PBS. After 90 min of incubation at room temperature, protected from light, the slides were washed and stained with DAPI (Cell Signaling, Danvers, MA, USA) at 1 μg/mL concentration in PBS, to visualize the nuclei. Slides were mounted with ProLong® Diamond Antifade Mountant (Life Technologies). Images were acquired using a Nikon Eclipse Ti-U fluorescence microscope and the NES Elements software.

The total protein was extracted from cells using RIPA lysis buffer, separated in SDS-PAGE, and transferred onto PVDF membranes. After blocking with 5% milk for 1 h, the membranes were probed with primary antibodies shaking at 4 °C overnight, followed by secondary antibody conjugation at room temperature for 1 h. The membranes were visualized using the ECL Western Blotting Detection system (GE Healthcare) or chemiluminescence western blot detection system (BioRad, Hercules, CA, USA). The following primary antibodies were used: anti-HER2 (C-18, SC-284) and anti-CPT2 (G5, SC-377294) antibodies were bought from Santa Cruze Biotechnology (Santa Cruze, CA, USA). anti-CPT1A (D3B3, Catalog# 12252), anti-OCT4 (C30A3, Catalog# 2840), anti-SOX2 (D6D9, Catalog# 3579) anti-NANOG (D73G4, Catalog# 4903), anti-Histone H3(D1H2, Catalog#4499), and anti-acetylated-Lysine (Catalog#9441) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-ACAD9 antibody was kindly gifted from Dr. Vockley Lab. Anti-CD47 (Catalog# PA2223) antibody were from Boster (Pleasanton, CA, USA). Anti-β-actin (Catalog# A2066) was from Sigma-Aldrich, anti-NF-κB p65 (acetyl K310) (Catalog# ab19870), and anti-Histone H3 (acetyl pan, Catalog# ab47916) antibodies were bought from Abcam (Cambridge, MA, USA).