Full moon Phospho-Explorer antibody array

Processing of the phospho-explorer array (PEX100) was performed following the Full Moon Biosystems guidelines. OPCs were immunopanned from wildtype mice and expanded in the presence of PDGF. Upon confluency, OPCs were differentiated through supplementation of CNTF, NT3 and T3. After 2 days of differentiation cells were starved for 4–5 hr in media devoid of supplementation to remove stimulation from the media and then treated for 15 min with either vehicle or BQ3020 (100 ng/mL). Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Calbiochem – 539134 and 524621) and processed for the array as recommended by Full Moon Biosystems.

Briefly, proteins present in the lysates were biotinylated through incubation with biotin. Chips were blocked using milk and biotinylated protein lysates were washed over. Binding of the individual proteins to each antibody spot on the array was assessed through fluorescent labelling with a dye-labelled streptavidin read using an Innopsys 710-IR scanner and analysed using Mapix software.

For each antibody the background intensity was subtracted, dye signal normalised and an average calculated of the duplicate spots. The ratio was calculated of binding to the phosphorylated amino acids vs the binding to the non-modified regions of the protein for each molecule, calculating this for both control and BQ3020 treated cells. The fold change in phosphorylation for each targeted amino acid was generated by comparing BQ3020 to vehicle. For selection a fold change of greater than two and less than 0.5 was set as the cut-off.

Antibody array was performed once – one cell lysate per condition.