Chemi reagent mix

Sera from CAR WT and CAR^HEPKO mice were used for cytokine array. The array was performed using Proteome Profiler Mouse Cytokine Array Panel A Kit (R&D systems #ARY006) according to the manufacturer’s instruction. Briefly, membranes were blocked for one hour at room temperature. Meanwhile, samples were incubated with detection antibody for one hour at room temperature. Then, the blocked membranes were incubated in the sample/detection antibody mixtures overnight at 4 °C. Next day, the membranes were washed three times and incubated with diluted Streptavidin-HRP for 30 min. at room temperature. After washing three times, the membranes were incubated with Chemi Reagent Mix and each spot was visualized on ChemiDoc™ touch imaging system (Bio-Rad). The signal intensities of each spot were calculated using ImageJ software, and each value was normalized against the mean values of reference spots (pre-defined by the manufacturer).

Analysis of 35 different apoptosis-related proteins was conducted in accordance with the manufacturer’s instructions. First, the arrays were incubated in 4-well multi-dish with a block buffer, Array Buffer 1. Then the buffer was removed and every array was incubated with the samples overnight at 4 °C on a rocking platform shaker. The unbound proteins were removed and the arrays were washed with 20 mL washing buffer three times. Detection Antibody Cocktail was added to all arrays for 1 h. The arrays were washed again with the washing buffer three times. A specific biotinylated detection antibody was added to all wells on a rocking platform shaker for 30 min. Then the spots were visualized by the Chemi Reagent Mix and evaluated by Bio-Rad Chemidoc XRS + system. The mean intensities of the spots were measured by Image Lab Software (BIO-RAD, USA).