Sc 136548

Expression of proteins was examined by western blotting. The standard procedures including SDS-polyacrylamide gel electrophoresis, membrane transference, antibodies incubation, and enhanced chemiluminescence detection were applied for 20 μg of total protein. The primary antibodies including CCL16 (ab199162, 1:2,000), B cell lymphoma (Bcl)-2 (ab196495, 1:2,000), Bcl-2-associated X protein (Bax; ab199677, 1:1,000), cleaved-caspase-3 (ab49822, 1:500), IL-6 (ab208113, 1:1,000), IL-1β (ab2105; 1:1,000), TNF-α (ab6671, 1:2,000), and GAPDH (ab8245, 1:10,000) were provided by Abcam (Cambridge, UK). Primary antibodies against TLR4 (sc-293072, 1:500) and phosphorylated P65 (p-P65; sc-136548, 1:500) and p-IκB-α (sc-8404, 1:500) were obtained from Santa Cruz (Shanghai, China). The polyvinylidene fluoride (Millipore, Billerica, MA, USA) membrane used for protein transfer was blocked with 5% non-fat milk at room temperature for 1 h. Then, the membranes were incubated with the aforementioned primary antibodies at 4°C overnight and reincubated with horseradish peroxidase-labeled secondary antibodies against Rabbit IgG (ab205718, 1:50,000; Abcam) and Mouse IgG (ab97023, 1:20,000; Abcam) at room temperature for 2 h. The relative protein levels were normalized to GAPDH and then compared to control.

The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam). The recombinant proteins used in the present study were as follows: human TIMP-3 (Cat No 973-TM-010, R&D Systems), human tumor necrosis factor-alpha (TNF-α) (Cat No 210-TA, R&D Systems), and human VEGF (Cat No 293-VE-050, R&D Systems).

Western blot analysis was performed as described previously (Liu et al., 2012 (link)). The primary antibodies against LC3B (dilution 1:1000, ab51520, abcam), SQSTM1 protein (dilution 1:1000, ab56416, abcam), p-NFκB (dilution 1:500, sc136548, santa cruz), MnSOD (dilution 1:5000, ab13533, abcam), and HRP-conjugated secondary goat antibodies (dilution 1:5000, SA00001-1 and SA00001-2; Proteintech) were used.

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo-Fisher Scientific). Equal amounts of protein were subjected to SDS-PAGE using 7.5% to 15% Tris-Glycine gradient gels, and blotted onto PVDF membranes (Bio-Rad Laboratories, Inc.; Hercules, CA, USA). After blocking with 5% skimmed milk, membranes were incubated with primary antibodies against IκBα (1:200), p-IκBα (1:200), HIF-2α (1:500), p65 (1:200, ab7970; Abcam; Burlingame, CA, USA), p- NF-κB p65 (Ser536) (1:200, sc-136548, Santa Cruz Biotechnology), MMP13 (1:200), ADAMTS5 (200:1), COL2 (2000:1, MAB8887; Merck Millipore), or β-ACTIN (1:1,000; Sigma-Aldrich) in Can Get Signal solution (Toyobo). The membranes were then incubated with HRP-conjugated secondary antibody (Promega; Fitchburg, WI, USA), and immunoreactive bands were visualized with ECL plus (GE Healthcare; Buckinghamshire, England, UK) according to the manufacturer’s instructions. The blots were stripped by incubating for 20 min in stripping buffer (2% SDS, 100 mM 2-mercaptoethanol, and 62.5 mM Tris-HCl, pH 6.7) at 50 °C and reblotted with other antibodies. Original images of the immunoblots were shown in Supplementary Fig. S4.