Ab211061

MDA-MB-231Luc2+GFP+ were harvested from the hind limbs of tumour-positive mice. Tibia and femurs were dissected, centrifuged to harvest bone marrow and crushed using a pestle and mortar. The single cell suspension of bone and bone marrow underwent red blood lysis using ammonium chloride (STEM cell technologies, Vancouver, BC, Canada). Lysed samples were stained with TOPRO-3 (Thermo Fisher, Waltham, MA, USA) to determine live cells. GFP+ tumour cells were identified using the FACSMelody (BD).
Once GFP+ tumour cells were isolated from vehicle and treated animals, cells were lysed according to the Human MAPK phosphokinase array instructions (Abcam, Cambridge, UK). Protein concentrations were measured using a BCA assay and protein blots conducted as per supplier’s instructions (abcam- ab211061). Blots were imaged using the Bio-Rad Gel Doc system and software (Image Lab version 6.0.1) to determine band intensities.

The human MAPK phosphorylation antibody array (ab211061; Abcam) was used due to manufacturer´s instructions. MiR-E-METAP1- or miR-E-METAP2-transduced MDAMB-TA or MCF7-TA cells were treated for 4 days with BKM at EC20 ± Dox. Chemiluminescent signal was detected using the Fusion SL Detection System (Vilber Lourmat) and FusionCapt Advance software (Vilber Lourmat). For analysis the ImageJ plugin Protein Array Analyser by Carpentier and Henault was used 131 . The average integrated area from 4 random background spots was subtracted from each antibody spot and then the signal was normalized to the average integrated area of the reference spots.