Journal: Breast Cancer Research : BCR

Article Title: Direct inhibition of PI3K in combination with dual HER2 inhibitors is required for optimal antitumor activity in HER2+ breast cancer cells

doi: 10.1186/bcr3601

Figure Lengend Snippet: PIK3CA mutation uncouples phosphoinositide 3-kinase signaling from HER2 inhibition by lapatinib. (A) BT474 and SKBR3 cells infected with wild-type, E545K or H1047R constructs were treated with lapatinib at the indicated doses, and lysates were analyzed by immunoblotting with the indicated antibodies. (B) Lysates from PIK3CA wild-type or mutant expressing cells treated with a range of lapatinib doses (0.0016 to 5 μM) were analyzed by ELISA for pHER2, pAkt and pS6. Half-maximal concentration (IC 50 ) values were calculated, and the mean log IC 50 ± SEM values for three replicate dose–inhibitor curves are shown. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. (C) HER2+ cell lines with wild-type PIK3CA (BT474 or SKBR3) or with a PIK3CA mutation (MDA361, HCC1954, SUM190 or UACC893) were treated with varying lapatinib doses and analyzed as described in (B) . Mean log IC 50 values from three replicates ± SEM are shown. Mean IC 50 data are shown in Table .

Article Snippet: Antibodies from the following sources were used for analysis: pHER2 Y1248 (R&D Systems, Minneapolis, MN, USA); Y877 pHER2 (Epitomics, Burlingame, CA, USA); Y1221/2 pHER2, Y11197 and Y1289 pHER3, S473 pAkt, Akt, S240/44 pS6, pErk1/2, Erk, and p110α PI3K (Cell Signaling Technology, Danvers, MA, USA); p85 N-terminal Src homology 2 (SH2) domain (EMD Millipore); HA (Covance, Princeton, NJ, USA); actin (Sigma-Aldrich, St Louis, MO, USA); HER2 (Thermo Fisher, Pittsburgh, PA, USA); and glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Mutagenesis, Inhibition, Infection, Construct, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Molecular Oncology

Article Title: Effects of trastuzumab and afatinib on kinase activity in gastric cancer cell lines

doi: 10.1002/1878-0261.12170

Figure Lengend Snippet: Kinetic and concentration‐dependent effects of trastuzumab and afatinib on the activation of EGFR and HER 2 in NCI ‐N87 cells. The levels of activated receptors were determined after treatment of NCI ‐N87 cells with trastuzumab and/or afatinib by western blot analysis. Activation of the receptors was analyzed using pEGFR ‐specific (Y1068) and pHER 2‐specific (Y1221/2) antibodies. (A) The levels of activated receptors were determined after 5 min of treatment of NCI ‐N87 cells with afatinib (0.01, 0.1, 0.5, 1 μ m ) and/or trastuzumab (5 μg·mL −1 ). (B) Cells were treated for 5, 20, 60, and 120 min with trastuzumab (5 μg·mL −1 ) and/or afatinib (0.5 μ m ). Equal loading of the lanes was confirmed by the detection of α‐tubulin or β‐actin. The depicted results are representative of three independent experiments. The results of the densitometric analysis and P ‐values are shown in the Supplement (Fig. , Fig. , Table , and Table ). A, afatinib; T, trastuzumab; Ctrl, control.

Article Snippet: The following antibodies were used: anti‐EGFR (Cell Signaling Technology (CST), distributed by New England Biolabs in Frankfurt, Germany; #2232; dilution 1 : 1000 in 5% BSA TBS‐T), anti‐pEGFR Y1068 (Life Technologies, Darmstadt, Germany; # 44788G; dilution 1 : 2000 in 5% milk TBS‐T), anti‐HER2 (Cell Signaling Technology (CST), distributed by New England Biolabs in Frankfurt, Germany; #2165; dilution 1 : 1000 in 5% BSA TBS‐T), anti‐pHER2 Y1221/2 (Cell Signaling Technology (CST), distributed by New England Biolabs in Frankfurt, Germany; # 2249; dilution 1 : 1000 in 5% BSA TBS‐T), anti‐pHER3 Y1289 (CST; # 4791; dilution 1 : 1000 in 5% BSA TBS‐T), anti‐α‐tubulin (Sigma‐Aldrich, Steinheim, Germany; # T9026; dilution 1 : 10 000 in 5% milk TBS‐T), anti‐β‐actin (Sigma‐Aldrich; A1978; dilution 1 : 5000 in 5% milk TBS‐T), anti‐mouse IgG (GE Healthcare, distributed by VWR in Ismaning, Germany; # NA931; dilution 1 : 10 000 in 5% milk TBS‐T), and anti‐rabbit IgG (CST; # 7074; dilution 1 : 2000 in TBS‐T).

Techniques: Concentration Assay, Activation Assay, Western Blot