Mouse anti hmgb1

Cells were collected in Cell Lysis Buffer, as usual in our lab (Vaz et al., 2015 (link)). Briefly, total cell extracts were lysed for 5 min on ice with shaking, collected with cell scrapper, and sonicated for 20 s. The lysate was then centrifuged at 14,000 g for 10 min at 4°C, and the supernatants were collected and stored at −80°C. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer's specifications. Then, equal amounts of protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% (w/v) nonfat milk solution, membranes were incubated with primary antibody mouse anti-HMGB1 (1:200, from Biolegend) diluted in 5% (w/v) BSA overnight at 4°C, followed by the secondary antibody goat anti-mouse HRP-linked (1:5,000, sc-2005, Santa Cruz Biotechnology®) diluted in blocking solution. Chemiluminescence detection was performed by using LumiGLO® reagent (Cell Signaling) and bands were visualized in the ChemiDoc^TM XRS System (Bio-Rad). The relative intensities of protein bands were analyzed using the Image Lab^TM analysis software (Bio-Rad).

Western blot analysis was performed as previously described [53 (link)] with minor alterations. Briefly, 100 μg of protein from tissue extracts were separated on a 12% SDS-PAGE gel. Following electrophoretic transfer onto a nitrocellulose membrane and blocking with 5% milk solution, the blots were incubated with primary antibody overnight at 4°C [rabbit anti-TLR4 (Santa Cruz, Dallas, TX, USA, #sc-10741; 1:500), mouse anti-HMGB1 (BioLegend, San Diego, CA, USA, #651402; 1:500), rabbit anti-ATX (Millipore, Billerica, MA, USA, #ABT28; 1:500), or mouse anti-β-actin (Sigma, USA, #A5441; 1:5,000)] and with horseradish peroxidase-labeled secondary antibody [anti-mouse or anti-rabbit (Santa Cruz, USA, #sc-2005 and #sc-2004, respectively; 1:5,000)] for 1 h at RT. Protein bands were detected by LumiGLO® (Cell Signaling, Danvers, MA, USA) and visualized by chemiluminescence with ChemiDoc (Bio-Rad, Hercules, CA, USA). Expression was quantified by computerized image analysis using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA, USA).