Acetyl α tubulin

LV tissue was homogenized in ice-cold lysis buffer containing PBS (pH 7.4), 0.5% Triton X-100, 300 mM NaCl, and protease/phosphatase inhibitor cocktail (Thermo Fisher) using the Next Advance Bullet Blender. Lysates were clarified at 16000×g for 5 min prior to protein concentration determination via BCA Protein Assay Kit (Pierce). Proteins were resolved by SDS/PAGE, transferred to nitrocellulose membranes (Bio-Rad), and membranes were blocked with 4% milk. Membranes were probed overnight with indicated primary antibodies for mouse monoclonal acetyl-lysine (Cell Signaling Technology; 9681), rabbit polyclonal acetyl-lysine (Cell Signaling Technology; 9441), acetyl-α-tubulin (Santa Cruz Biotechnology; sc-23950), α-tubulin (Santa Cruz Biotechnology; sc-23948), or total histone H3 (Cell Signaling Technology; 4499). Horseradish peroxidase (HRP)–conjugated secondary antibodies (Southern Biotech) were used at a concentration of 1:2000. SuperSignal West Pico chemiluminescence system (Thermo Scientific) and a ChemiDoc XRS+ imager (Bio-Rad) were used to detect protein expression.

Extraction and concentration determination of sample protein were performed as previously described^{41 (link)}. Equal amount of protein from different samples was loaded on SDS polyacrylamide gels, then were transferred to nitrocellulose membrane. After blocking with Tris-buffered saline (TBS) containing 5% fat-free milk, the blots were incubated with primary antibodies overnight at 4 ℃ and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, CA, USA) for 1 h at room temperature. Rabbit antibodies against p-AKT (473), p-AKT (308), AKT, and p70S6K, and mouse antibody against p-p70S6K were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against EGFR, HER2, HER3, acetyl-α-tubulin, cyclin D, cyclin E, CDK2, CDK4, CDK6, and actin were purchased from Santa Cruz (USA). Antibodies against p-HER2 (Tyr1248) and p-HER3 (Tyr1289) were purchased from Affinity (China). Antibodies against p-EGFR (Tyr 1069) and α-tubulin were purchased from Abscience (China). Subsequently, the blots were washed with TBST for three times, incubated with ECL reagent (Millipore, USA), and visualized by ChemiDoc XRS+ gel imaging system (Bio-Rad, USA). Image gray value analysis was performed by ImageJ (NIH, USA) and the intensities of actin were used as a control for all other bands^{40 (link)}.

HBEC were harvested with 1X cell lysis buffer (Cell Signaling Technology, Danvers, MA) with protease inhibitors (cOmplete mini, Roche, Indianapolis, IN, USA). Vascular homogenates were generated as described in the brain microvascular isolation methods section. Capillary electrophoresis immunoblots were performed using the ProteinSimple Wes system (San Jose, CA, USA). The following primary antibodies were used: phospho-eNOS S1176/1177 (BD Biosciences, #612392, San Jose, CA, USA), phospho-eNOS T495 (BD Biosciences, #612706, San Jose, CA, USA), eNOS (Millipore, 07-520, Billerica, MA), phospho-tau T231 (AT180, Thermo Scientific, MN1040, Waltham, MA, USA), β-actin (Cell Signaling Technology, 4970 S, Danvers, MA, USA), acetyl-α-tubulin (Santa Cruz Biotechnology, sc-23950, Dallas, TX, USA), and α-tubulin (Cell Signaling Technology, 2144 S, Danvers, MA, USA). Data was analyzed using the Compass for SW software (ProteinSimple, San Jose, CA, USA). Chemiluminescent signals were measured as area under the curve. Additional antibody information is supplied in Supplementary Table 1.