y984  (Cell Signaling Technology Inc)

Journal: Oncotarget

Article Title: Store operated calcium entry is altered by the inhibition of receptors tyrosine kinase

doi: 10.18632/oncotarget.24685

Figure Lengend Snippet: (A) Immunoblot analysis of phospho-EGFR Y1068 , total EGFR, phospho-ErbB2 Y1248 and total ErbB2 in SK-BR-3 cells. The same protocol than in Figure was used, SK-BR-3 were treated at 37°C for 10 minutes with erlotinib 5μM or a vehicle. Immunoblots presented in A are representative of at least three independent experiments. Lanes separated by a thin line have been cropped from the same immunoblot. (B) Relative quantification of immunoblots presented in A and C. EGFR phosphorylation obtained after erlotinib 5μM or gefitinib 1μM treatment was compared to total EGFR and then normalized to the control condition. Results are expressed as means ± S.D. of at least three independent experiments, Student’s t-test, non significant. (C) Immunoblot analysis of phospho-EGFR Y1068 , total EGFR, phospho-ErbB2 Y1248 and total ErbB2 in SK-BR-3 cells. SK-BR-3 cells were treated 10 min with gefitinib. Immunoblots presented in C are representative of at least three independent experiments. Lanes separated by a thin line have been cropped from the same immunoblot. (D) Relative quantification of immunoblots presented in A and C. ErbB2 phosphorylation obtained after erlotinib 5μM or gefitinib 1μM treatment was compared to total ErbB2 and then normalized to the control condition. Results are expressed as means ± S.D of at least three independent experiments, Student’s t-test, * P < 0.05. (E) Immunoblot analysis of phospho-ErbB3 Y1289 and phospho-ErbB4 Y984 in SK-BR-3 cells. SK-BR-3 cells were treated 10 min with lapatinib 2μM, CP-724714 2μM, trastuzumab 1μM, erlotinib 5μM or the vehicle. Immunoblots presented in E are representative of two independent experiments. β-actin was used as a loading control.

Article Snippet: The blots were saturated with TBS-T buffer (0.02 M Tris(hydroxymethyl)aminomethane, 0.138 M NaCl, 0.05% Tween 20, pH 7.6), containing 5% BSA or milk for 1 h at room temperature, incubated overnight at 4 °C with primary antibodies: anti-EGFR, anti-phospho-EGFR Y1068 , anti-ErbB2, anti-phospho-ErbB2 Y1248 , anti-ErbB3, anti-phospho-ErbB3 Y1289 , anti-phospho-ErbB4 Y984 , anti-Akt, anti-phospho-Akt S473 , anti-ERK1/2, anti-phospho-ERK1/2, GAPDH (1:1000, Cell Signaling Technology), and anti-β-actin (1:10,000, Sigma-Aldrich).

Techniques: Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: A Pre-Clinical Assessment of the Pan-ERBB Inhibitor Dacomitinib in Pediatric and Adult Brain Tumors

doi: 10.1016/j.neo.2018.02.004

Figure Lengend Snippet: Dacomitinib reduces ERBB4 phosphorylation in vivo but antagonizes conventional medulloblastoma chemotherapeutics in vitro . (A) SMO mice harboring medulloblastomas were treated with vehicle, dacomitinib (doses indicated) or a combination of dacomitinib and cyclophosphamide (CPA, 150 mg/kg). Tumors were stained for phosphorylated or total ErbB4, stain intensity determined using ImageJ, and the ratio of phosphorylated:total ErbB4 was determined. Shown is mean ± SEM, n = 5 mice per group. Groups were compared using a Student’s t-test (* P < .05). In vitro interactions between dacomitinib and ( B ) 4-hydroperoxycyclophosphamide (4HPC), ( C ) cisplatin and ( D ) vincristine were determined using CompuSyn. Graphs show the combination index (CI) values versus factor affected (Fa) values, where values above or below the dashed line indicate antagonism or synergism, respectively. Mean CI ± standard deviation are shown. Closed circles, open squares and open triangles indicate replicate experiments.

Article Snippet: Tissue sections (5 μm) underwent microwave antigen retrieval in a citrate buffer before immunostaining with the following antibodies: Ki-67 (Leica #NCL-Ki67p, 1:5000), cleaved caspase-3 (CC3) (BD #559565, 1:500), p-EGFR Y1173 (Cell Signaling #4407, 1:250), phosphorylated ErbB4 (pErbB4) Y984 (Cell Signaling #3790, 1:50), or total ErbB4 (Santa Cruz #SC-283, 1:200).

Techniques: Phospho-proteomics, In Vivo, In Vitro, Staining, Standard Deviation