Phospho explorer antibody array

The specific humanin-mediated signaling pathways identified using the Phospho Explorer Antibody Array (Full moon Biosystems, Sunnyvale, CA, USA) were analyzed using Ingenuity^® Pathway Analysis (IPA; QIAGEN, Redwood city, CA, USA). A spreadsheet containing the list of humanin-mediated phosphorylated proteins was generated by using Partek^® Genomomics Suite (Partek Incorporated, St. Louis, Missouri, USA) and was uploaded into IPA. The criteria for the selection of the proteins of interest were an absolute fold change > 1.2 between control and HNG treated groups and P < 0.05. The software mapped each of the proteins to the repository of information in the Ingenuity Pathways Knowledge base. Molecular networks and canonical pathways regulated by humanin were obtained using IPA core analysis. Category rankings were based on the p values derived from the Fisher's exact test and the cut-off threshold for significance was a P-value < .05. Ratios were calculated as follows: number of genes in a given pathway that meet the cutoff criteria, divided by the total number of genes that make up that pathway and that are in the reference gene set.

The phosphorylation profiling (Additional file 1: Table S1) of decorin-treated and non-treated mouse primary neurons was determined by Phospho Explorer Antibody Array (Full Moon BioSystems, Cat. PEX100) following the manufacturer’s protocol. The alterations of phospho-protein levels in decorin-treated/non-treated with ratios > 1.5 or < 0.6 were subjected to gene ontology enrichment analysis [17 (link)–19 (link)]. The biological processes with false discovery rate < 0.01 were considered as significantly enriched.

For comparison of the activation and expression of phosphoproteins by rMSA treatment, AML12 hepatocytes were treated with or without rMSA (600 μM) for 24 h. Then, the samples were treated with cell lysis buffer (Full Moon BioSystems, USA). Next, phosphatase inhibitor and protease inhibitor were added to each sample at a volume ratio of 1:50, and each sample was treated with a tube of Full Moon cell lysis beads. After the above steps, the proteins of the samples were extracted, and then, Phospho Explorer Antibody Array (Full Moon BioSystems, USA) was used to react with the protein according to the standard procedures provided by Full Moon. The array contained 1318 antibodies, and each of them had 2 replicates. Wayen Biotechnologies (Shanghai) performed the experiment and analyzed the data. The extent of protein phosphorylation was calculated by the following equation: $$\text{phosphorylation}\text{ratio}=\text{phosphorylated}\text{value}/\text{unphosphorylated}\text{value}.$$

Neural stem cells were differentiated under 4 W/kg RF-EMF exposure for 48 h. The changes of signaling pathways were detected by a Phospho Explorer Antibody Array (Full Moon Biosystems, CSP100^plus, United States). Data collection and analysis were carried out by Wayen Biotechnologies (Shanghai, China). Briefly, 452 proteins in 16 signaling pathways were analyzed. The analyzed results were first normalized by housekeeping protein as phosphorylation-protein/housekeeping and total protein-expression/housekeeping. The ratio of protein expression and phosphorylation change after RF-EMF exposure was obtained by comparing it to the control.

Hybridization and analysis of the Phospho Explorer Antibody Array were carried out by Full Moon BioSystems (Sunnyvale, CA, USA) and served to survey the phosphorylation state of select proteins. The assay was performed using BMDMs pretreated for 60 min with 10 μM SB747651A, followed by LPS+Dex for 4 h. At the end of the incubation time, proteins were collected and analysed. Physical and functional association among the downregulated genes after MSK1 inhibition was done by using the STRING 10.1 database and the network was visualized using Cytoscape 3.1.0.