Anti α tubulin

Organs were disrupted in extraction buffer (20 mM HEPES, 350 mM NaCl, 2 mM EDTA pH 8.0, 10% glycerol, 0.1% Tween 20, 1 mM PMSF, 1x cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich), 1 M DTT) using a POLYTRON homogenizer (KINEMATICA, Luzern, Switzerland) followed by three cycles of freezing and thawing. Extracts were incubated for 1 hr at 4°C with Benzonase Nuclease (125 U/ml; Merck Millipore, Darmstadt, Germany) to release nuclear proteins. 100 μg of whole cell protein extracts were separated using NuPAGE Novex^TM 3–8% Tris-Acetate protein gels (Thermo Fisher Scientific, Schwerte, Germany) for 4 hr (80 V). After overnight transfer (8 V) to PVDF membranes, blots were probed with primary antibodies in blocking solution (2% milk powder/1% BSA in 0.05% Tween 20/PBS): rabbit polyclonal anti-hSET1B (1:500; A302-281A BETHYL Laboratories, Montgomery, TX) and rabbit monoclonal anti-α-tubulin (1:2000; 1878–1 Epitomics, Burlingame, CA). Following incubation with the goat anti-rabbit secondary IgG HRP antibody (1:50,000; 31460 Thermo Fisher Scientific), chemiluminescence was detected on an ImageQuant LAS 3000 system (GE Healthcare, Munich, Germany).

To assess mTOR protein levels in the sciatic nerve, we dissected nerves from the sciatic notch to just proximal to the trifurcation. These nerve segments were flash-frozen in liquid nitrogen, cut into small pieces with microdissection scissors, and homogenized in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Na₂EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na₃VO₄, 1 μg/ml leupeptin) with phosphatase inhibitor mixtures 1 and 2 (Invitrogen, ThermoFisher Scientific, Waltham, MA). Equal protein amounts (15 μg) were loaded and analyzed by SDS-PAGE and western blot. Antibodies used were anti-mTOR (1:1000; Cell Signaling Technologies, Danvers, MA) and anti-α-tubulin (1:1000; Abcam, Cambridge, MA). Western blot images were quantified using FIJI. All bands were normalized to background and mTOR bands were compared with α-tubulin levels.

Total proteins from the treated retinal endothelial cells were extracted with RIPA lysis buffer (Beyotime; cat.: P0013C) containing PMSF (1:100; Beyotime; cat.: ST506). The protein concentration was quantified by BCA assay (Sangon biotech; cat.: C503061) according to the manufacturer’s instruction, and further adjusted to 2 mg/ml before dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). When the SDS-PAGE gel electrophoresis was completed, the proteins were transferred onto a nitrocellulose filter membrane (NC; Millipore; cat.: HATF00010). After blocking with 5% milk in PBST buffer for 1 h, the membrane were incubated with the primary antibodies anti-STAT3 (1:1000; Cell Signaling Technology; cat.: 9139S), anti-p-STAT3 (1:1000; Cell Signaling Technology; cat.: 9145 T), anti-HIF-1α antibody (1:1000; Proteintech; cat.: 20,960-1-AP), anti-VEGFA (1:1000; Abcam; cat.: ab1316) and anti-α-tubulin (1:8000; Abcam; cat.: ab18207) at 4°C overnight. After that, the membrane was incubated with the corresponding secondary antibody conjugated with HRP (1:1000; Beyotime; cat.: A0208) for 2 h at room temperature. After rinsing with PBST buffer for three times, the immunoreactivity of membrane was visualized using the SuperSignal West Pico kit (Pierce) [20 (link)].

Cells were incubated and lysed with ice-cold RIPA buffer containing complete protease inhibitors and phosphatase inhibitor cocktail. Protein concentrations were quantified with a BCA protein assay kit (Thermo Scientific). All samples were diluted into equal protein concentration. Western blot analysis was performed as previously described 36 . Primary antibodies included anti-STING (Cat# 13674, Cell Signaling Technology, 1:1000), anti-CD68 (Cat# ab955, Abcam, 1:1000), anti-GAPDH (Cat# ab181602, Abcam, 1:10000), anti-phospho-STAT1 (Cat# 9167, Cell Signaling Technology, 1:1000), anti-STAT1 (Cat# 14994, Cell Signaling Technology, 1:1000), anti-phospho-STAT3 (Cat# 9145, Cell Signaling Technology, 1:1000), anti-STAT3 (Cat# 12640, Cell Signaling Technology, 1:1000), anti-Vinculin (Cat# ab129002, Abcam, 1:10000), anti-β-actin (Cat# 3700, Cell Signaling Technology), and anti-α-tubulin (Cat# ab7291, Abcam, 1:5000). The bands were visualized by enhanced chemiluminescence (Millipore).

The proteins from the hippocampus were separated using a 10-12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C; the primary antibodies included anti-CRMP2 (1:20,000; Abcam, ab129082); anti-pCRMP2 (Thr514 site) (7 μl: 5 ml; Abcam, ab85934); anti-DNMT1 (1:5,000); anti-DNMT3a (1:5,000); anti-DNMT3b (1:5,000); anti-α-tubulin (1:5,000; Abcam, ab7291); anti-Tyr-Tubulin (1:2,500; Sigma, T9028); anti-Acet-Tubulin (1:200; Santa Cruz Biochemistry, sc-23950); and anti-GAPDH (1:5,000; Beyotime, AF1186). After washing three times, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime, 1:5,000) at room temperature for 1 h. Then, the membranes were visualized by an ECL chemiluminescent detection kit (Beyotime, P0018S), and the intensities of the protein bands were calculated by ImageJ software. The relative expression of the proteins was normalized to that of GAPDH.

AMs or lung tissues were washed thrice with cold phosphate buffer saline (PBS) and homogenized in RIPA buffer with protease and phosphatase inhibitor mixture (Roche Diagnostics) for 30 min on ice. After centrifugation for 10 min at 12,000 rpm at 4°C, the supernatant was collected. 30μg of protein was separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Millipore, US) and hybridized using standard procedures. The blots were probed with anti-HO-1 (diluted 1: 1,000, Abcam, MA, USA), anti-Cav-1 (diluted 1:1,000, Abcam, MA, USA), anti-IκB(diluted 1:1,000, Cell Signaling Technology, Inc.)and anti-α-Tubulin (diluted 1:1,000, Abcam, MA, USA) antibodies. After incubation with Horseradish Peroxidase- conjugated to IgG secondary antibodies (diluted 1:5000, Boster), the relative signal intensity of bands was determined and standardized by chemiluminescence imaging system ChemiDoc (Bio-Rad) and the Image Lab software (Bio-Rad). Protein relative expression was normalized to α-Tubulin.