Journal: British Journal of Cancer

Article Title: Defining the molecular response to trastuzumab, pertuzumab and combination therapy in ovarian cancer

doi: 10.1038/bjc.2012.176

Figure Lengend Snippet: HER receptor signalling. Phosphorylation of human epidermal growth factor receptors (HERs) 1, 2 and 3 after 14 days of therapy and control as detected by western blotting ( A ). Anti-HER2 C-terminal western blotting using day 14 tumours showed two bands, p185 and p95, but anti-extracellular domain (ECD) of HER2 blotting revealed another band (p110) consistent with the ECD produced by shedding from full-length HER2 ( B ). Anti-ECD and anti-C-terminal antibodies after 14 days in whole tissue sections using quantitative immunofluorescence ( C ). The ECD to C-terminus ratio ( D ) was increased in trastuzumab-treated groups (alone or combined), suggesting that increased shedding inhibited by trastuzumab.

Article Snippet: Antibodies used for western blotting were as follows: anti-phosphorylated HER1 (pHER1) (Tyr 992 ; Cell Signaling Technology, Danvers, MA, USA) at 1 : 1000; anti-phosphorylated HER2 (pHER2) (Tyr 877 ; Cell Signaling Technology) at 1 : 1000; anti-phosphorylated HER3 (pHER3) (Tyr 1289 ; Cell Signaling Technology) at 1 : 1000 and anti-actin (Merck, Whitehouse Station, NJ, USA).

Techniques: Phospho-proteomics, Control, Western Blot, Produced, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth *

doi: 10.1074/jbc.M109.098301

Figure Lengend Snippet: Analyses of tyrosine phosphorylation of ErbB-2 in different LNCaP and MDA PCa2b cells. Upper panels, A, LNCaP C-33 and C-81 cells were seeded in regular culture medium at a density of 5 × 105/T25 flask for 3 days. p indicates phosphorylation. B, MDA PCa2b cells (passages 38 and 89) were seeded with a density of 1 × 106/T25 flask in regular culture medium. Cells were fed with fresh medium and cultured for another 2 days. Immunoblottings were performed with Abs to different tyrosine phosphorylation sites of ErbB-2. After stripping, the membrane was hybridized with Ab to detect ErbB-2 protein. Similar results were obtained from over three sets of independent experiments. Lower panels, growth rates of different LNCaP and MDA PCa2b cells. A, LNCaP cells were seeded at a density of 3 × 104 cells/well in 6-well plates in the regular culture medium. 3 days after plating, one set of attached cells was harvested and counted as day 0. The remaining cells were refreshed with the regular medium, and the total cell numbers were counted on the indicated days. B, for MDA PCa2b cell growth kinetics, cells were seeded in regular medium at a density of 1 × 105 cells/well in 6-well plates and counted on days 3, 5, and 7. Data shown are the ratios of cell numbers normalized to the corresponding number on day 0. Bars = the range of duplicates. Similar results were obtained from at least three sets of independent experiments. Y, tyrosine.

Article Snippet: Anti-phospho-HER-2/ErbB-2 (Tyr 877 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1112 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1221/1222 ) Ab, anti-phospho-STAT3 (Tyr 705 ) Ab, anti-phospho-STAT5 (Tyr 694 ) Ab, anti-phospho-Src (Tyr 416 ) Ab, anti-phospho-Akt (Ser 473 ) Ab, anti-STAT3 Ab, anti-STAT5 Ab, and anti-Src Ab were from Cell Signaling Technology (Beverly, MA).

Techniques: Cell Culture, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth *

doi: 10.1074/jbc.M109.098301

Figure Lengend Snippet: Co-immunoprecipitation of cPAcP and ErbB-2. A, confluent C-33 cells were maintained in a steroid-reduced medium for 48 h. Cells were then harvested, lysed, and immunoprecipitated by PAcP Ab (ATM-3) (36). The immunoprecipitate was electrophoresed and blotted (IB) with anti-ErbB-2 Ab (C-terminal (C-Ter) Ab) and anti-PAcP Ab (Sigma catalog number P-9808). B, C-33 cells in a steroid-reduced medium for 48 h were harvested and lysed for immunoprecipitation by ErbB-2 Ab (N-terminal (N-Ter) Ab). The immunoprecipitate was immunoblotted with anti-PAcP Ab (ATM-3) and anti-ErbB-2 Ab (C-terminal Ab). Similar results were obtained from three sets of independent experiments. C, LNCaP C-33 cells were seeded at a density of 0.6 × 106/T75 (Subconfluent) and 4.5 × 106/T75 (Confluent) for 3 days and then maintained in steroid-reduced medium for 2 days. Cells were harvested and separated into membrane (Mem), cytoplasmic (Cyto), and nuclear (Nu) protein subfractions using the subcellular protein fractionation kit. Immunoblottings were performed with respective Abs to detect ErbB-2, cPAcP, Shc proteins, EGFR, PYK2, and HNF3α. The intensity of the hybridization band was semiquantified and calculated for a total of 100% for each group. EGFR, PYK2, and HNF3α served as markers for the respective subcellular fractionation. D and E, confluent LNCaP C-33 cells were harvested after being maintaining in a steroid-reduced medium for 2 days. The total cell lysates, membrane, and cytoplasmic fractions, separated by the subcellular protein fractionation kit, were immunoprecipitated by cPAcP Ab (ATM-3) or ErbB-2 Ab (N-terminal Ab). The immunoprecipitate was immunoblotted with anti-PAcP Ab (Santa Cruz Biotechnology (Santa), sc-80908) and anti-ErbB-2 Ab (C-terminal Ab). Similar results were obtained from three sets of independent experiments.

Article Snippet: Anti-phospho-HER-2/ErbB-2 (Tyr 877 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1112 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1221/1222 ) Ab, anti-phospho-STAT3 (Tyr 705 ) Ab, anti-phospho-STAT5 (Tyr 694 ) Ab, anti-phospho-Src (Tyr 416 ) Ab, anti-phospho-Akt (Ser 473 ) Ab, anti-STAT3 Ab, anti-STAT5 Ab, and anti-Src Ab were from Cell Signaling Technology (Beverly, MA).

Techniques: Immunoprecipitation, Fractionation, Hybridization

Journal: The Journal of Biological Chemistry

Article Title: Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth *

doi: 10.1074/jbc.M109.098301

Figure Lengend Snippet: Effect of cPAcP expression on tyrosine phosphorylation of ErbB-2 in LNCaP C-81 cells. A, LNCaP C-81 cells were transiently transfected with WT PAcP, mutant PAcP H12A cDNAs, or vector alone. Cells were harvested after 48 and 72 h, respectively, and an equal amount of cellular lysates was separated by SDS-PAGE for Western blot analyses. cPAcP, the total and the specific tyrosine phosphorylation (p) level of ErbB-2, and phosphorylation of p52Shc and PCNA were detected by the corresponding Ab. The protein levels of ErbB-2, Shc, and β-actin were examined with the respective Ab after the membranes were stripped. B, cPAcP, ErbB-2, and total tyrosine phosphorylation of ErbB-2 protein in WT PAcP cDNA-transfected LNCaP-28 and LNCaP-40 stable sublines were detected with anti-PAcP, anti-ErbB-2, and 4G10 anti-Tyr-phosphorylation Abs, respectively. For cell growth kinetics, cPAcP-28, cPAcP-40, and LNCaP C-81 cells were seeded at a density of 5 × 104 cells/well in 6-well plates in regular medium for 3 days. One set of attached cells was harvested and counted as day 0. The remaining cells were fed with regular medium. Cells were harvested on days 2, 4, and 7 for counting. Similar results were obtained in two sets of independent experiments in duplicate wells. *, p < 0.05 versus LNCaP C-81 cells; **, p < 0.01 versus LNCaP C-81 cells.

Article Snippet: Anti-phospho-HER-2/ErbB-2 (Tyr 877 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1112 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1221/1222 ) Ab, anti-phospho-STAT3 (Tyr 705 ) Ab, anti-phospho-STAT5 (Tyr 694 ) Ab, anti-phospho-Src (Tyr 416 ) Ab, anti-phospho-Akt (Ser 473 ) Ab, anti-STAT3 Ab, anti-STAT5 Ab, and anti-Src Ab were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth *

doi: 10.1074/jbc.M109.098301

Figure Lengend Snippet: Effect of transient knockdown of cPAcP by siRNA in LNCaP C-33 cells on tyrosine phosphorylation of ErbB-2. A, lysate proteins of transiently transfected cells with different amounts of PAcP siRNA-126 plasmids for 48 h were analyzed for cPAcP, cyclin D1, PCNA, and the phosphorylation (p) levels of ErbB-2, p52Shc, ERK1/2, Akt, Src, STAT3, and STAT5 proteins. After stripping the membrane, the protein levels of ErbB-2, Shc, ERK1/2, Akt, Src, STAT3, STAT5, and α-tubulin were detected, respectively. Similar results were obtained from five sets of independent experiments. B, LNCaP C-33 cells were plated at a cell density of 3 × 105/well in 6-well plates for 48 h and then transfected with different amounts of PAcP siRNA-126 plasmids. Control cells were transfected with the vector containing scramble oligonucleotides. Cell numbers were counted 3 days after transfection. The ratio of cell growth was calculated by normalizing the cell number to that of the control. The result shown is the average from three sets of independent experiments in duplicates (n = 2 × 3). Bars = S.E.

Article Snippet: Anti-phospho-HER-2/ErbB-2 (Tyr 877 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1112 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1221/1222 ) Ab, anti-phospho-STAT3 (Tyr 705 ) Ab, anti-phospho-STAT5 (Tyr 694 ) Ab, anti-phospho-Src (Tyr 416 ) Ab, anti-phospho-Akt (Ser 473 ) Ab, anti-STAT3 Ab, anti-STAT5 Ab, and anti-Src Ab were from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, Stripping Membranes, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth *

doi: 10.1074/jbc.M109.098301

Figure Lengend Snippet: Kinetic effects of knockdown of cPAcP on tyrosine phosphorylation of ErbB-2. LNCaP C-33 cells were transiently transfected with siPAcP-126 plasmid or vector alone and harvested at 30, 36, and 48 h after transfection. An equal amount of cellular lysates was separated by SDS-PAGE for Western blot analyses. cPAcP, the specific tyrosine phosphorylation (p) level of ErbB-2, and phosphorylation of p52Shc were detected by the corresponding Ab. The protein levels of ErbB-2, Shc, and α-tubulin were examined after the membranes were stripped. Similar results were obtained from three sets of independent experiments.

Article Snippet: Anti-phospho-HER-2/ErbB-2 (Tyr 877 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1112 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1221/1222 ) Ab, anti-phospho-STAT3 (Tyr 705 ) Ab, anti-phospho-STAT5 (Tyr 694 ) Ab, anti-phospho-Src (Tyr 416 ) Ab, anti-phospho-Akt (Ser 473 ) Ab, anti-STAT3 Ab, anti-STAT5 Ab, and anti-Src Ab were from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth *

doi: 10.1074/jbc.M109.098301

Figure Lengend Snippet: Tyrosyl phosphorylation of ErbB-2 protein and the growth properties of PAcP knockdown stable subclones. A, siPAcP stable sublines were seeded in regular culture medium with 5 × 105/T25 flask for 3 days. Total lysates from PAcP siRNA-transfected stable subclone cells (C-11, C-3, C-17) and the control subclone cells (V-3) were analyzed by Western blotting. The data shown are representative of three sets of independent experiments. B, for growth analyses, cells were seeded in duplicates at a density of 5 × 104/well in 6-well plates. After 3 days, the numbers of each stable subclone cell were counted at the time points as indicated in the figure. The relative cell growth was represented by the ratio of total cell number normalized to the respective number on day 0. Similar results were obtained from three sets of independent experiments (n = 2 × 3). Bar = S.E. C, Cells were seeded in duplicates in the condition as described above. After 4 days of culturing in steroid-reduced medium, the cell number was counted. The relative cell growth was represented by the ratio of total cell number normalized to that of V-3 control cells. Similar results were obtained from three sets of independent experiments (n = 2 × 3). Bar = S.E. Inset, cPAcP protein was analyzed by Western blotting from one set of experiments. D, xenograft animal model for siPAcP subclone cells. 1 × 106 cells of each subline in 0.1 ml of medium with 0.1 ml of Matrigel were injected into five female nude mice/group. The tumor growth was monitored weekly. *, p < 0.05 versus V-3 control cells.

Article Snippet: Anti-phospho-HER-2/ErbB-2 (Tyr 877 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1112 ) Ab, anti-phospho-HER-2/ErbB-2 (Tyr 1221/1222 ) Ab, anti-phospho-STAT3 (Tyr 705 ) Ab, anti-phospho-STAT5 (Tyr 694 ) Ab, anti-phospho-Src (Tyr 416 ) Ab, anti-phospho-Akt (Ser 473 ) Ab, anti-STAT3 Ab, anti-STAT5 Ab, and anti-Src Ab were from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, Western Blot, Animal Model, Injection