Ab189860

Protein samples were resolved by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis for 2 h and electrophoretically transmitted to a PVDF membrane (Merck Millipore, USA). After blocking nonspecific binding sites with 5% skim milk for 60 min at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against SIRT3 (1 : 1000; ab189860), Bax (1 : 1000; ab32503), Bcl-2 (1 : 1000; ab59348), ALP (1 : 1000; ab95462), Osterix (1 : 500; ab22552), OCN (1 : 500; ab93876), β-actin (1 : 1000; ab8227), SOD2 (1 : 5000; ab13533), and Ac-SOD2 (1 : 5000; ab137037) (all from Abcam, Cambridge, UK). Then, the membranes were rinsed in Tris-buffered saline with Tween 20 and incubated with the corresponding secondary horseradish peroxidase-conjugated antibodies (1 : 1000) for 2 h at room temperature. The proteins were detected using chemiluminescent HRP substrates (Millipore Corporation, Billerica, MA, USA).

Total proteins from the exosomes or cells were extracted and detected using the bicinchoninic acid kit. Briefly, 40 µg of total protein was separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (120 V, 90 min) and transferred to polyvinylidene fluoride (PVDF) membranes (90 V, 90 min). For blocking, 5% non-fat milk was added to the PVDF membranes for 1 h. Anti-CD9 (1: 1,000, ab307085, Abcam, Cambridge, MA, USA), anti-CD63 (1: 1,000, ab217345, ab68418), anti-CD81 (1: 1,000, ab109201), anti-SIRT3 (1: 500, ab189860), anti-AMPK (1: 1,000, ab207442), anti-p-AMPK (1: 1,000, ab133448), anti-LC3II/I (1: 1,000, ab48394), anti-P62 (1: 1,000, ab240635), anti-NLRP3 (1: 1,000, ab263899), anti-apoptosis-associated speck-like protein containing a CARD domain (ASC) (1: 1,000, ab70627), anti-IL-1β (1: 1,000, ab283822), anti-iNOS (1:1,000, ab178945), anti-IL-6 (1: 1,000, ab233706), anti-TNF-α (1:1,000, ab183218), anti-Calnexin (1:1000, ab22595) and anti-GAPDH (1:1,000, ab8245) were added and incubated overnight at 4 °C. Moreover, the horseradish peroxidase-labeled goat-anti-rabbit secondary antibody (1:5,000 diluted) was incubated at 25 ℃ for 2 h. Protein blots were detected using Pierce™ ECL (Thermo Fisher, Waltham, USA) in ChemiDoc MP (Bio-Rad, California, USA).

Protein expression was examined by Western blot as previously described [34 (link)]. HPMECs or lung tissues were lysed using RIPA buffer, and protein concentration was examined using the Pierce BCA protein assay kit (23227, Thermo Fisher Scientific, USA). The polyvinylidene difluoride (PVDF) membranes were purchased from GE Healthcare (10600023, USA). The following primary antibodies were employed: VE-cadherin (1 : 1000, ab33168, Abcam, USA), β-catenin (1 : 1000, 610154, BD Transduction Laboratories, USA), dephosphorylated active β-catenin (05-665, Millipore, Germany), Sirt3 (1 : 500, ab189860, Abcam, USA), matrix metallopeptidase-7 (MMP-7, 1 : 400, ab5706; Abcam, USA), and cyclooxygenase-2 (COX-2, 1 : 1000, ab62331, Abcam, USA). α-Tubulin was purchased from the Proteintech Company (Hubei, China). The secondary antibodies of goat antirabbit (1 : 5000, ab6721; Abcam, USA) or goat antimouse (1 : 5000, A21010; Abbkine, USA) were used. PVDF membranes were visualized using a chemiluminescence Western blotting detection reagent. The signal intensity of each immunoblot was analyzed using ImageJ software (version 1.48v; NIH, USA), and each band density was normalized by the α-tubulin protein expression level. To clearly present the results, the value of protein expression in the sham-operated or control group was further normalized to 1 using the mean.