Ab67282

Samples for the identification of PTMs were prepared according to the approach described earlier [82 (link),83 (link)]. Proteins extracted from the thymus were separated with Laemmli SDS PAGE [80 (link)] (Figure S2, Supplementary Materials). The isolated proteins were identified with Western blot using antibodies ab 79823, ab 67282 (Abcam, Cambridge, UK) against HMGB1 and HMGB2, respectively. The strips cut from the polyacrylamide gel corresponding to the HMGB1 and HMGB2 proteins were treated with a solution of 40% acetonitrile in 0.1 M ammonium bicarbonate at 37 °C for 15 min, followed by enzymatic trypsin hydrolysis (4 h, 37 °C) directly in the gel. The trypsinolysis reaction was stopped by adding 0.5% and 10% of trifluoroacetic acid and acetonitrile, respectively. Samples for MALDI mass spectrometry were obtained by co-crystallizing a solution of the trypsinolysis products with a solution containing 20 mg/mL of 2,5-dihydroxybenzoic acid in 30% acetonitrile, 0.1% trifluoroacetic acid, followed by air-drying.

Brain tissues or PMNs on cytospin slides were washed twice with cold PBS and lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 0.5% Triton X-100, 0.5% NP-40, 0.25% sodium-deoxycholate, 150 mM NaCl, and complete Mini protease inhibitor cocktail tablets (1 tablet in 10 ml) (Roche, Basel, Switzerland). Lysates were centrifuged for 15 min at 14000 rpm at 4 °C and supernatants were loaded into 10–12% SDS PAGE gels. Primary antibodies for anti-CitH3 (ab18956–100; Abcam, Cambridge, UK), anti-HMGB1 (ab67282; Abcam, Cambridge, UK), and anti-GAPDH (2118; Cell Signaling Technology, Danvers, MA) were diluted 1:2000–10,000. The signals were detected using a chemiluminescence kit (Merck Millipore, Darmstadt, Germany).

A pre‐cooled RIPA lysis buffer (Sigma) was applied to lyse the experimental cell lines. A bicinchoninic acid protein (BCA) assay kit (Sigma) was used to detect the total protein concentration. SDS‐PAGE loading buffer (5X) was mixed with 10 µg total proteins and heated at 100°C for 5 minutes. Thereafter, SDS‐PAGE (4%‐12%) was used to separate proteins. The proteins were subsequently transferred to PVDF membranes (0.45 µm, EMD Millipore) and placed in 4°C for 2 hours. Next, 5% non‐fat milk was used to block the PVDF membranes at room temperature for 1 hour. After incubation with primary antibodies at 4°C overnight, the PVDF membranes were treated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (1:20 000; Southern Biotech) placed in 37°C for 1 hour. Immobilon Western Chemiluminescent HRP substrate (EMD Millipore) was used to visualize the protein bands. Primary antibodies (Abcam) were as follows: anti‐HMGB2 (1:500, ab67282), anti‐cleaved caspase‐3 (1:500, ab2302), anti‐p53 (1:500, ab32389) and anti‐β‐actin (1:1000, ab8227).