Journal: Cancer Biology & Therapy

Article Title: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

doi: 10.4161/cbt.29171

Figure Lengend Snippet: Figure 1. Trastuzumab induces phosphorylation of ErbB2-Y1248 and ErbB1-Y845, inhibits Akt phosphorylation and downregulates ErbB3 in trastuzumab-sensitive SKBR3 and BT474 cells. (A) SKBR3 cells (2 × 106) were plated in 10 cm dishes, serum-starved overnight and treated either with trastuzumab (4 μg/mL) or EGF (100 ng/mL) for the indicated times or left untreated. After harvesting, the whole cell lysates (WCL) were incubated with RayBio human EGFR phosphorylation antibody array 1 according to the instructions provided by the manufacturer. The pair of dots in red rectangles indicates phosphorylated ErbB1-Y845 (ErbB1-pY845); blue rectangles indicate ErbB2-pY1248; purple rectangles indicate ErbB2-pY1112. The map of EGFR phosphorylation antibody array 1 is available at http://www.raybiotech.com/. (B) Changes in phosphorylation, as detected by RayBio human EGFR phosphorylation antibody array 1, following treatment of BT474 cells with trastuzumab or EGF for the indicated times. The experimental procedures were essentially the same as described under (A). Red rectangles indicate phosphorylation signal for ErbB1-Y845; blue rectangles indicate phosphorylation signal for ErbB2-pY1248. Black rectangles indicate phosphorylation signal for ErbB2-pT686 and pS1113 (from left to right). The experiments with RayBio human EGFR phosphorylation antibody array 1 were repeated at least twice for both cell lines. (C) SKBR3 cells were serum-starved and incubated either with trastuzumab or EGF for the indicated times. The cells were harvested and the levels of ErbB2-pY1248, ErbB1-pY845, P-Akt-T308, and P-Akt-S473 or P-ERK1/2 and total levels of the indicated proteins in WCL were determined by western blot analysis. Actin western blot analysis of WCL was done to control for equal loading (note: from here on, actin western blots were used to control for equal loading in all figures). Bottom two panels, levels of ErbB1-pY845 and total ErbB1 were determined in ErbB1 immunoprecipitate by western blot analysis. (D) Western blot analysis of BT474 cells treated either with trastuzumab or EGF. The experimental procedures were essentially the same as (C). (E) SKBR3 and BT474 cells were serum-starved overnight and treated with trastuzumab for 1 h or left untreated. The levels of phosphorylated ErbB3-Y1289 (ErbB3-pY1289) and total ErbB3 were detected by western blot analysis using antibodies directed against ErbB3-pY1289 or ErbB3. (F) The cells were grown in the media supplemented with 10% FBS and then treated with trastuzumab (10 μg/mL) for the indicated times. WCL harvested from indicated cells were subjected to western blot analysis. The levels of ErbB3 in WCL were detected using an antibody directed against ErbB3.

Article Snippet: RayBio Human EGFR phosphorylation antibody array 1 can detect 17 phosphorylation sites of ErbB family of receptor tyrosine kinases.

Techniques: Incubation, Ab Array, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

doi: 10.4161/cbt.29171

Figure Lengend Snippet: Figure 2. Trastuzumab activates ErbB2 tyrosine kinase and induces ErbB2-Y1248 phosphorylation in the presence of lapatinib. (A) Increased ErbB1-Y845 phosphorylation was detected following trastuzumab treatment of SKBR3 cells. Serum-starved SKBR3 cells were treated for 1 h. After harvesting the WCL, the immunoprecipitation reaction with antibody recognizing ErbB2 (29D8) was performed to detect the heterodimer between ErbB1 and ErbB2. (B) The experiments were performed similar to those described in (A) except that prior to harvesting the WCL, trastuzumab-treated BT474 cells were crosslinked with DTSSP reagent according to the modified protocol provided in the literature.5 (C) Trastuzumab induces the activation of ErbB2 kinase activity in BT474 and SKBR3 cells. After serum-starving overnight, SKBR3 and BT474 cells were either treated with trastuzumab for 1 h or EGF for 15 min, or left untreated as indicated. WCL were harvested and subjected to immunoprecipitation using either human control IgG or trastuzumab. ErbB2 tyrosine kinase activity in each immunoprecipitate was determined using a universal tyrosine kinase assay kit (Takara Bio Inc.) according to manufacturer’s instructions. Data are expressed as mean ± SEM. Statistical significance was determined by the Student t test. *P < 0.05. (D) SKBR3 cells were plated and grown in the serum-containing media and then serum-starved overnight. Cells were either pre-treated with lapatinib (200 nM) for 4 h or not pretreated and then either treated with trastuzumab (4 μg/mL) or left untreated. Analysis of phosphorylation levels of ErbB family phosphorylation sites was done by RayBio human EGFR phosphorylation antibody array 1 according to the manufacturer’s instructions. (E) Analysis of phosphorylation level in ErbB family receptors following treatment of BT474 cells with trastuzumab, lapatinib, or trastuzumab plus lapatinib. The experimental procedures were essentially the same as those described in (D).

Article Snippet: RayBio Human EGFR phosphorylation antibody array 1 can detect 17 phosphorylation sites of ErbB family of receptor tyrosine kinases.

Techniques: Immunoprecipitation, Modification, Activation Assay, Activity Assay, Universal Tyrosine Kinase Assay, Ab Array