Horse anti mouse hrp conjugate

Rabbit anti-nestin (for immunofluorescence 1:2500, for western blot 1:2000; BioLegend (San Diego, CA, USA, 839801), mouse anti-GFAP (for immunofluorescence 1:100; Merck (Darmstadt, Germany), MAB360; for western blot 1:250; Dako (Glostrup, Denmark), M0761), chicken anti-vimentin (for immunofluorescence 1:1000; used throughout the study; for western blot 1:2000; BioLegend, 919101), rabbit anti-vimentin (1:200; Abcam (Cambridge, UK), ab45939; used for the comparison in Figure 1), rabbit anti-TOMM20 (1:200; Abcam, ab186734), mouse anti-Ki67 (1:50, BD Biosciences (Franklin Lakes, NJ, USA, 550609), goat anti-chicken Alexa Fluor 488 (1:1000; Thermo Fisher Scientific, (Waltham, MA, USA, A11039), donkey anti-mouse Alexa Fluor 555 (1:1000; Thermo Fisher Scientific, A31570), donkey anti-rabbit Alexa Fluor 647 (1:1000; Thermo Fisher Scientific, A31573), donkey anti-rabbit Alexa Fluor 555 (1:1000; Thermo Fisher Scientific, A31572), rabbit anti-GAPDH–HRP conjugate (1:500; Cell Signaling Technology, (Beverly, MA, USA, 3683), goat anti-rabbit-HRP conjugate (1:1000; Cell Signaling Technology, 7074), and horse anti-mouse-HRP conjugate (1:1000; Cell Signaling, 7076) were used. The specificity of the GFAP, vimentin, and nestin antibodies was previously validated, on tissues/cell cultures from mice carrying null mutations in the respective genes serving as negative controls.

Twenty‐five microliters of cell‐free supernatant or 25 mg of total protein cell lysate was resolved on a 4–12% NUPAGE™ pre‐cast SDS‐polyacrylamide gel electrophoresis (PAGE) gel (Life Technologies Inc.) at 90 V (15 min) then 180 V (60 min). Transfer of immobilized proteins to nitrocellulose membrane was carried out at 100 V for 1 h and the membrane was blocked in Tris‐buffered saline‐tween containing 10% dried‐milk powder for 1 h at room temperature. Membranes were probed with rabbit anti‐human high‐mobility group box 1 (HMGB1) primary antibody (1:5000 dilution, overnight; Abcam Ltd) and goat anti‐rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)‐conjugate secondary antibody (1:10 000 dilution, 2 h; Sigma‐Aldrich).
Streptavidin‐tagged RIPK3 and Bak were probed with strepMAB‐classic mouse anti‐human antibody (1:4000; IBA Life Sciences) and horse anti‐mouse HRP‐conjugate (1:10 000 dilution; Cell Signaling Technologies). Details of all other primary and secondary antibodies used are given in Supporting Information Table S3. Membranes were developed and visualized using enhanced chemiluminescent substrate (ECL) and a Chemidoc Touch Imaging System (both Bio‐Rad). Image densitometric analysis was undertaken using Image J software.