Pex100

The phosphorylation explorer antibody microarray chip PEX100, containing 1,318 well-characterized site-specific antibodies, was purchased from Full Moon Biosystems (Sunnyvale, CA, USA). Details of these antibodies can be accessed via the vendors website (http://www.fullmoonbio.com/product/phospho-explorer-antibody-array/). KYSE30 cells (2 × 10⁷ cells per 10-cm plate) were treated with or without 10 μM XMD8-92 for 2 h. Cells were washed with cold 1X PBS (4 °C) and collected by scraping from the plate. Cells were spun briefly in a microcentrifuge to remove culture media from the cells. Three additional washes were performed using cold 1X PBS (4 °C). Cells were centrifuged again at 4 °C and supernatant discarded. Cell pellets were shipped to the vendor and antibody microarray’s run according to the manufacturer’s instruction. Duplicate chips were run for both control and treated lysates groups and ‘% of control’ values calculated according to normalised average plate readings.

We used a phospho-specific protein microarray (PEX100, Full Moon BioSystems, USA) containing 1,318 antibodies against 584 phosphorylation sites of 432 proteins for phosphorylation profiling. The antibody array experiment was performed by Wayen Biotechnology (Shanghai, China) according to an established protocol. The SureScan Dx Microarray Scanner was used to scan for chip images, and the raw data were acquired using GenePix Pro v6.0 software. The phosphorylation ratio of each protein was calculated as follows: phosphorylation ratio = phospho value/unphospho value.

The protein expression and phosphorylation data were generated using the PEX100 (Full Moon BioSystems, Inc, Sunnyvale, CA, USA). HBMSCs were cultured with control medium or Si (BG extracts diluted to 1/128) for 7 days. Then wash the cells with pre‐chilled PBS and add the cell lysis solution dedicated to the Full Moon chip to lyse the cells. Add a cell lysis magnetic beads dedicated to the Full Moon chip to each sample, shake at high speed for 1 minute, then stand on ice for 10 minutes and repeat the process 4 times. Centrifuge the sample at 4°C, 15000 g, 15 minutes and transfer the supernatant to a new centrifuge tube. Centrifuge the supernatant again and repeat the process four times. After the last centrifugation, transfer the supernatant to a new tube and stored at −20°C before they were subject to the assessment of the protein chip. The chip analysis was done by Wayen Biotechnologies (Shanghai, China), Inc, and signalling pathway enrichment was performed by KEGG.