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. Author manuscript; available in PMC: 2021 Sep 15.
Published in final edited form as: Clin Cancer Res. 2021 Jan 7;27(6):1681–1694. doi: 10.1158/1078-0432.CCR-20-3017

Figure 2. Effects of neratinib combination on downstream signaling in HER2+ cells.

Figure 2.

(A) Combination of neratinib with everolimus, trametinib, alpelisib and sapanisertib. HCC-1954, BT-474 and Sk-BR-3 cells were incubated with single drug or combination drugs for 24 hours. Treatment groups include (1) vehicle; (2) neratinib at 1 μM; (3) everolimus at 2 nM; (4) trametinib at 0.2 μM; (5) alpelisb at 1 μM; and (6) sapanisertib at 0.2 μM; or (7) neratinib + everolimus; (8) neratinib + trametinib; (9) neratinib + alpelisb; and (10) neratinib + sapanisertib at the same concentrations as single treatment. (B) Combination of neratinib with palbociclib. Neratinib was used at 10, 25, and 250 nM on SK-BR-3, BT-474, and HCC-1954 cell lines respectively. Palbociclib was used at 25 μM on all three cell lines. Cell lysates were analyzed by western blot. Phosphorylated protein levels of the key signaling molecules in the MAPK and PI3K/Akt pathways, including phospho-Akt, phospho-4E-BP1, phospho-S6K, phospho-S6, phospho-ERK1/2, phospho-MEK1/2 and phospho-Rb, were detected by their specific antibodies. Their total protein levels and β-actin levels were also detected for controls.