Horseradish peroxidase conjugated donkey anti goat antibody

Cells were lysed in M-PER™ Mammalian Protein Extraction Reagent (78501; Thermo Scientific) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Applied Science, Mannheim Germany). Protein homogenates were separated under denaturing conditions on Any-KD or 10% SDS-polyacrylamide gels (Mini-PROTEAN Precast gels; Bio-Rad, Hercules, CA) and electro-blotted on to PVDF membranes. For pro-IL-1β detection, the membranes were blocked in Superblock T20 (Thermo Fisher Scientific) and incubated with 0.1 μg/ml goat anti-mouse IL-1β antibody (AF-401-NA; R&D Systems) diluted in 20% Superblock T20/TBST, and subsequently a horseradish peroxidase-conjugated donkey anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Other membranes were blocked with 5% dry milk/TBST and proteins were incubated with 5% dry milk/TBST or 5% BSA/TBST according to manufacturer's protocol. Protein expression was detected by chemiluminescence (SuperSignal West Pico; Thermo Fisher Scientific). Protein quantifications were done with the ImageJ software.

Total protein homogenates of LV from sham operated mice and from mice with compensated and decompensated hypertrophy due to aortic banding-induced pressure overload. Briefly, LV tissue was homogenized in T-PER Tissue protein extraction reagent (Thermo Scientific, Rockford; IL) supplemented with protease and phosphate inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail; Thermo Scientific). The homogenates were cleared by centrifugation and concentrations were measured using the BCA assay (Bio-Rad, Hercules, CA). Protein homogenates were separated under denaturing conditions on 4–20% SDS-polyacrylamide gels (Mini-PROTEAN TGX Precast gels) and electroblotted on to PVDF membranes (Thermo Scientific). The membranes were blocked in Superblock T20/TBS (Thermo Scientific) and incubated with 0.1 µg/ml (diluted in Superblock T20/TBS) goat anti-mouse CCL21 antibody (AF457; R&D Systems) and subsequently a horseradish peroxidase-conjugated donkey anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA). CCL21 expression was detected by chemiluminescence (SuperSignal West Pico; Thermo Scientific) and the C-Digit Blot Scanner (Li-Cor). The membranes were stripped using Restore Western Blot Stripping buffer (Thermo Scientific), blocked and reprobed with 0.05 µg/ml mouse anti-GAPDH antibody (G8795, Sigma-Aldrich). Densitometrical quantification was performed using ImageJ (NIH).