antibodies specific human phospho-p130cas y410 or y165  (Cell Signaling Technology Inc)

Journal: International Journal of Clinical and Experimental Medicine

Article Title: Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites

doi:

Figure Lengend Snippet: Primer pairs for target and housekeeping genes used in qRT-PCR assays

Article Snippet: Cell lysates were resolved by standard SDS-PAGE and electroblotted onto a PVDF membrane and incubated with rabbit anti human integrin β1 (Abcam, Cambridge, MA) and rabbit anti human p130Cas antibodies (Cell Signaling Technology, Inc., Danvers, MA) and with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugate antibody (CWbiotech Inc., Beijing, China).

Techniques:

Journal: International Journal of Clinical and Experimental Medicine

Article Title: Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites

doi:

Figure Lengend Snippet: Effects of mechanical stress on target gene expressions in stretched upper back and medial arm skin fibroblasts. At each time points of mechanical loads, individual cultures of fibroblasts were collected and assayed for relative levels of gene expression of integrin β1 (Panel A), p130Cas (Panel B), TGF β1 (Panel C) and type I collagen (Panel D). Data are shown as the mean ± SD based on triplicate experiments. * and †P<0.05 versus medial arm fibroblasts stressed for 36 and 48 hours, respectively.

Article Snippet: Cell lysates were resolved by standard SDS-PAGE and electroblotted onto a PVDF membrane and incubated with rabbit anti human integrin β1 (Abcam, Cambridge, MA) and rabbit anti human p130Cas antibodies (Cell Signaling Technology, Inc., Danvers, MA) and with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugate antibody (CWbiotech Inc., Beijing, China).

Techniques: Gene Expression

Journal: International Journal of Clinical and Experimental Medicine

Article Title: Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites

doi:

Figure Lengend Snippet: Effects of mechanical stress on target protein productions in stretched upper back and medial arm fibroblasts. At each time point following mechanical loads, each culture of fibroblasts was collected and used for Western-blot detection of integrin β1 and p130Cas (Panel A) and afterward semi-quantified and normalized to that of internal reference of GAPDH respectively (Panel B, C); or the conditioned supernatant from each group for ELISA assay to detect protein contents of TGF β1 and type I collagen (Panel D and Panel E, respectively). Data are shown as the mean ± SD based on triplicate experiments. * and †P<0.05 versus medial arm fibroblasts stressed for 36 and 48 hours, respectively. M: Medial side of upper arm; S: Scapular part of upper back skin.

Article Snippet: Cell lysates were resolved by standard SDS-PAGE and electroblotted onto a PVDF membrane and incubated with rabbit anti human integrin β1 (Abcam, Cambridge, MA) and rabbit anti human p130Cas antibodies (Cell Signaling Technology, Inc., Danvers, MA) and with a secondary anti-rabbit horseradish peroxidase (HRP)-conjugate antibody (CWbiotech Inc., Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: Association of clinicopathologic variables with p130cas staining *

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Staining

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: p130Cas expression in human epithelial ovarian carcinoma. A) Representative images of low vs high levels of immunohistochemical peroxidase staining for p130cas. Both are shown at ×200 magnification (scale bar = 50 μm). The negative control (×100) represents a sample of ovarian cancer tissue used in this study that was processed by immunohistochemistry with the secondary antibody alone. B) Kaplan–Meier curves of overall survival of patients whose ovarian tumors expressed high vs low levels of p130cas. Two-sided comparisons were made using the log-rank statistic. Survival probabilities and 95% confidence intervals at 2 and 4 years for low p130cas expression were 95% (95% CI = 91% to 99%) and 80% (95% CI = 65% to 95%), respectively, and for high p130cas expression were 53% (95% CI = 42% to 65%) and 15% (95% CI = 3% to 27%), respectively. C) Kaplan–Meier curves of progression-free survival for patients whose ovarian tumors expressed high vs low levels of p130cas. Two-sided comparisons were made using the log-rank statistic. Survival probabilities and 95% confidence intervals at 2 and 4 years for low p130cas expression were 47% (95% CI = 34% to 60%) and 7% (95% CI = 2% to 12%), respectively, and for high p130cas expression were 13% (95% CI = 7% to 18%) and 3% (95% CI = 1% to 5%), respectively.

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control, Immunohistochemistry

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: Multivariable analysis of associations between clinicopathologic variables and disease-specific survival in ovarian cancer patients *

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Expressing

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: Silencing of p130cas gene expression with small interfering RNA (siRNA) in vitro and in vivo. A) Immunoblot analysis of p130cas protein expression in a panel of ovarian cancer cell lines. Equal amounts of total protein were loaded in each lane; β-actin was used as a loading control. B) Silencing of p130cas expression using siRNA. HeyA8 ovarian cancer cells were transfected with control siRNA or p130cas siRNA in vitro and analyzed after 48–72 hours for p130cas protein expression. β-actin was used as a loading control. C) Decreased p130cas protein expression in HeyA8 orthotopic ovarian tumors collected at the completion of a therapy experiment with p130cas siRNA in neutral nanoliposomes (siRNA-DOPC). β-actin was used as a loading control. D–F) p130cas siRNA-based therapies in nude mice injected with HeyA8 (D), SKOV3ip1 (E), or taxane-resistant HeyA8-MDR ovarian cancer cells (F). In each experiment, 10 mice per group were randomly assigned to receive therapy with control siRNA-DOPC, control siRNA-DOPC plus docetaxel, p130cas siRNA-DOPC, or p130cas siRNA-DOPC plus docetaxel. Mice were killed by cervical dislocation when the mice in the control group became moribund (HeyA8: 28 days; SKOV3ip1: 38 days; HeyA8-MDR: 32 days), and tumor weight and disease location were recorded. Mean tumor weight with 95% confidence intervals (left panels) and corresponding tumor weight distributions for each mouse model (right panels) are shown. Statistical analysis for tumor weights was performed by two-sided Mann–Whitney rank sum tests. Asterisks represent a statistically significant difference from the control siRNA-DOPC treatment group. P values for each comparison are listed above the brackets shown (for HeyA8: p130cas siRNA, P = .04; control siRNA-DOPC + docetaxel, P < .001; p130cas siRNA-DOPC + docetaxel, P < .001; for SKOV3ip1: control siRNA-DOPC + docetaxel, P = .02; p130cas siRNA-DOPC + docetaxel, P < .001; for HeyA8-MDR: p130cas siRNA + docetaxel, P = .002). Double dagger designates a statistically significant difference in tumor weights for mice treated with p130cas siRNA-DOPC plus docetaxel compared with control siRNA-DOPC plus docetaxel (SKOV3ip1, P < .001).

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Expressing, Small Interfering RNA, In Vitro, In Vivo, Western Blot, Transfection, Injection, MANN-WHITNEY

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: Orthotopic tumor development in mice treated with control or p130cas siRNA with or without docetaxel *

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques:

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: p130Cas signaling detected using RNA microarray. A) Statistically significant differences in gene expression in Hey8 cells treated with p130cas small interfering RNA (siRNA) vs control siRNA. HeyA8 treated with p130cas siRNA vs control siRNA in vitro were harvested and RNA was isolated. Genes were selected by univariate tests (two-sided two-sample t tests, P < .001). Six hundred forty-five genes were differentially expressed between control siRNA– and p130cas siRNA–treated cells. The data are presented in matrix format in which rows represent individual genes and columns represent each sample. Each cell in the matrix represents the expression level of a gene feature in an individual sample. The red and green colors reflect high and low expression levels, respectively, as indicated in the scale bar (log2 transformed scale). B) Summary of RNA microarray results. The data revealed an abrogation in cell death pathways, specifically, a statistically significant decrease in phosphatidylinositol-3 kinase catalytic subunit (PI3KCA) levels (two-sided two-sample t test, P = .046). SI = small interfering.

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Microarray, Expressing, Small Interfering RNA, In Vitro, Isolation, Transformation Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: Effect of p130cas small interfering RNA in neutral nanoliposomes (siRNA-DOPC) therapy on tumor cell apoptosis. A) Tumor cell apoptosis was assessed by immunofluorescent staining for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in tumors obtained from the SKOV3ip1 therapy experiment. Photomicrographs (×200 magnification) represent TUNEL-positive tumor cells (green) and tumor cell nuclei (blue); scale bar = 50 μm. The bar graph (right panel) shows the mean number of TUNEL-positive cells for each treatment group. The mean number of TUNEL-positive cells was calculated by averaging the TUNEL-positive cells from five random fields per slide (each slide represented a tumor from one mouse); five slides per treatment group were examined. Error bars represent 95% confidence intervals. Comparison between groups was performed by two-sided Mann–Whitney rank sum tests. Asterisks represent statistically significant increases in tumor cell apoptosis compared with siRNA-DOPC controls (p130cas siRNA-DOPC: P < .001; p130cas siRNA-DOPC + docetaxel: P < .001). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays at various time points after treatment with p130cas siRNA and/or docetaxel. Mean values for the measured optical density (OD) at 570 nm for 12 replicates are shown. Error bars represent 95% confidence intervals. B) SKOV3ip1 cell viability was statistically significantly decreased following p130cas siRNA (P < .001). C) SKOV3TRip2 cell viability was statistically significantly decreased following p130cas siRNA (P = .01, two-sided Mann–Whitney rank sum tests). Cell death was assessed with Annexin V-phycoerythrin (Annexin V-PE) and 7-aminoactinomycin D (7AAD) by fluorescence-activated cell sorting analysis at various time points after treatment with p130cas siRNA and/or docetaxel. Mean values for the three replicates with 95% confidence intervals are shown. Error bars represent standard error of the mean. D) Graphical representation of Annexin V-PE and 7AAD apoptosis detection assay. SKOV3ip1 ovarian cancer cells were either left untreated or treated with p130cas siRNA and/or docetaxel for 48, 72, or 96 hours, and the percentage of apoptotic cells was determined. Values are the means of three replicates and 95% confidence intervals. Focusing on the 72-hour time point, when p130cas expression is at its lowest, the brackets represent comparisons made by a two-sided Mann–Whitney rank sum test. Asterisks represent statistically significant increases in tumor cell apoptosis compared with the untreated group (among Annexin V-PE [+] samples, p130cas siRNA: P < .001; docetaxel: P < .001; p130cas siRNA + docetaxel: P < .001; among Annexin V-PE/7AAD [+] samples, p130cas siRNA: P < .001; docetaxel: P < .001; p130cas siRNA + docetaxel: P < .001) Double dagger represents statistically significantly increased tumor cell apoptosis (Annexin V-PE/7-AAD) with p130cas siRNA plus docetaxel compared with docetaxel therapy alone (P = .01; two-sided Mann–Whitney rank sum tests). E) Graphical representation of Annexin V-PE apoptosis detection assay with benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD-fmk) pretreatment. SKOV3ip1 cells were left untreated or subjected to p130cas siRNA and/or ZVAD-fmk pretreatment for 72 hours, and the percentage of apoptotic cells was then determined. The values shown are the means of three replicates and 95% confidence intervals. Comparisons between treatment groups were performed by Mann–Whitney rank sum test (two-sided). Asterisks represent statistically significant increases in tumor cell apoptosis compared with the untreated group (among Annexin V-PE [+] samples, p130cas siRNA: P = .002 two-sided Mann–Whitney rank sum tests). No difference in percent apoptosis was visualized with ZVAD-fmk pretreatment. F) Graphical representation of tumor cell migration. HeyA8 cells were either untreated or subjected to control or p130cas siRNA, and the percentage of migratory cells per high-powered field (HPF) was determined. There was statistically significantly decreased HeyA8 tumor cell migration with p130Cas siRNA. Comparisons between treatment groups were performed by two-sided Mann–Whitney rank sum tests. The asterisk represents a statistically significant decrease in tumor cell migration compared with the untreated group (p130cas siRNA: P < .001). G) Graphical representation of tumor cell invasion. HeyA8 cells were either left untreated or subjected to control siRNA or p130cas siRNA, and the percentage of invasive cells per HPF was determined. HeyA8 tumor cell invasion was statistically significantly decreased in the presence of p130Cas siRNA. Comparisons between treatment groups were performed by two-sided Mann–Whitney rank sum tests. The asterisk represents a statistically significant decrease in tumor cell invasion compared with the untreated group (p130cas siRNA: P < .001).

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Small Interfering RNA, Staining, End Labeling, TUNEL Assay, MANN-WHITNEY, Fluorescence, FACS, Detection Assay, Expressing, Migration

Journal: JNCI Journal of the National Cancer Institute

Article Title: Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

doi: 10.1093/jnci/djr372

Figure Lengend Snippet: Effect of p130cas small interfering RNA (siRNA) on tumor cell autophagy. A) Immunoblot analysis of cleaved poly-ADP ribose polymerase (PARP) at 72 hours following treatment of SKOV3ip1 and SKOV3TR cells with p130cas siRNA and/or docetaxel. Immunoblotting for total actin was performed to confirm equal loading. B) Immunoblot analysis of the Bcl-2 family members following treatment SKOV3ip1 and SKOV3TR cells with p130cas siRNA. Immunoblotting for total actin was performed to confirm equal loading of total protein. Densitometry was performed to objectively assess potential differences. When estimating relative amounts of phosphoproteins, equal loading was ensured by immunoblotting to determine amounts of the corresponding total proteins C) Immunoblot analysis of phosphorylated phosphatidylinositol-3-kinase (PI3K), total PI3K, phosphorylated Akt, total Akt, phosphorylated mammalian target of rapamycin (mTOR), and total mTOR following treatment with p130cas siRNA. When estimating relative amounts of phosphoproteins, equal loading of was ensured by immunoblotting to determine amounts of the corresponding total proteins. D) Co-immunoprecipitation and immunoblot analysis of Beclin-1 and Bcl-2, respectively, following p130cas silencing in SKOV3ip1 and SKOV3TR cells. Numbers below blots indicated densitometry for differences in Bcl-2 relative to Beclin-1. E) Immunoblot analysis for cleavage of LC3 and Beclin-1 following treatment with p130cas siRNA. Immunoblotting for total actin was performed to confirm equal loading. Densitometry results show relative expression of LC3-II and Beclin-1 each normalized to actin. F) Detection of autophagolysosomes by transfection with green fluorescent protein (GFP)–light chain 3 (LC3) following treatment with p130cas siRNA. SKOV3ip1 cells were transfected with GFP-LC3 plasmid and then treated with 1 μg of control siRNA or p130cas siRNA, Control siRNA–treated cells showed a diffuse expression pattern of GFP-LC3; however, autophagosomes formed in p130cas siRNA–treated cells. In fluorescence microscope images (×200 magnification), LC3 II had translocated and accumulated in the membranes of autophagosomes, signifying the induction of autophagy. About 200 cells were counted for each case and if more than five punctate pattern containing cells were present, the sample was considered to be positive for autophagy (scale bar = 50 μm). G) Detection of autophagolysosomes by transmission electron microscopy. SKOV3ip1 cells were grown on six-well plates, treated with p130cas siRNA or control siRNA or left untreated for 72 hours, fixed for 2 hours with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), fixed again in 1% OsO4 in the same buffer, and then subjected to the electron microscopic analysis (×4000 and ×20 000 magnification; scale bars = 10 μm [left panels] and 2 μm [right panels]). Representative areas were chosen for ultrathin sectioning.

Article Snippet: Twenty micrograms of whole-cell lysate was fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, blocked with 5% nonfat milk for 1 hour at room temperature, and probed with mouse anti-human p130cas antibody (Neomarkers) at 1:3000 dilution overnight at 4°C.

Techniques: Small Interfering RNA, Western Blot, Immunoprecipitation, Expressing, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy