Array vision evaluation 8

The Human AKT Pathway Phosphorylation Array kit (Raybiotech, Guangzhou, China) was used to detect the relative levels of phosphorylation of 18 AKT pathway proteins^{61 (link)}. The name and position for all 18 AKT pathway proteins are listed in Supplementary Data 4. In brief, the membrane was incubated with blocking buffer at room temperature for 30 min. The blocking buffer was discarded, and the protein sample was added to the well for complete incubation at 4 °C overnight. The membrane was washed using Wash Buffer I and Wash Buffer II and incubated with Detection Antibody Cocktail at 4 °C overnight. The membrane was washed and incubated with HRP-anti-rabbit IgG at room temperature for 2 h. The membrane was then incubated with the detection buffer mixture at room temperature for 2 min. Finally, the membrane was photographed under a chemiluminescence imaging system. Data collection was performed on Array Vision Evaluation 8.0 (GE, MA, USA). The relative fold change of the protein on two different arrays was calculated according to the formula in the user manual. The differential protein was determined according to the manufacturer’s criteria: (1) mean signal density > 150; (2) fold change ≤0.83 or ≥1.2.

Blood was harvested from 8-week-old wild-type or EμBmi1 transgenic mice and serum was isolated by centrifugation at 3000 rpm for 5 min. Serum protein arrays were performed using Biotin Label-based Mouse Antibody Array I (Raybiotech) according to the manufacturer’s instructions. Briefly, the arrays were blocked, incubated with 100 μL serum samples overnight, then incubated with biotin-conjugated antibodies (1/250) for 2 hours and with HRP-linked secondary antibody (1/1000) overnight. The membranes were incubated with chemiluminescent substrate and exposed to X-ray film for 15 minutes before development. Quantitative array analysis was performed using Array Vision Evaluation 8.0 (GE Healthcare Life Science). Cluster analysis (hierarchical clustering) of the standard value of the pass volcano plot (differential protein expression) was performed and sample proteins of close relationship were clustered together.

Serum samples were screened in duplicates using a Mouse Cytokine Array QAM-INF-1 (RayBiotech) containing slides coated with 40 different cytokines according to the manufacturer’s guidelines with some modifications as previously described [42 (link)]. Briefly, the arrays were blocked, incubated with 100 mL of condition medium overnight, followed by biotin-conjugated antibodies (1/250) incubation for 2 h and with HRP-linked secondary antibody (1/1000) for 1 h. The membranes were incubated with a peroxidase substrate, and the results were documented using XAR films. Quantitative array analysis was performed using Array Vision Evaluation 8.0 (GE Healthcare Life Science).