Tumor-associated 3D matrix induced Akt/PKB activity in MDA-MB-231 is regulated by both PI3K and beta-1 integrin. a. Representative Western blot showing levels of pAktS473 (pAkt), total Akt (tAkt), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as endogenous protein loading control, in lysates from MCF10A, MCF7 and MDA-MB231 cultured on 2D or within staged 3D ECMs. Specific Akt/PKB activity was calculated as the ratio of the scanned optical density (O.D.) of pAktS473/totalAkt (p/tAkt O.D.). The calculated activity ratios were normalized to ratios obtained for each cell line cultured on 2D control conditions and were individually assigned as one arbitrary unit. Note that tumor-associated 3D matrix induces high specific Akt/PKB activity levels in MDA-MB-231 but not in MCF-10A or MCF-7 cells. b. Lysates from MDA-MB-231 cells cultured on 2D or in staged 3D ECMs in the presence and/or absence of 10 and 50 nM Wortmannin (Wt10 and Wt50) and/or 50 μg/ml mAb13 (mAb13) were proved by Western immunoblotting using antibodies against pFAKY397 (pFAK), total FAK (tFAK), pAKTS473 (pAkt), total Akt (tAKT) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The percentages of inhibition of FAK (%inh FAK) and Akt/PKB (%inh Akt) activities are shown and were calculated using the corresponding O.D. ratios: pFAKY397/totalFAK and pAktS473/totalAkt. Note that while Wortmannin inhibited Akt/PKB activity, mAb13, alone or in combination with Wortmannin, inhibited both FAK and Akt/PKB activities.