Journal: International Journal of Proteomics

Article Title: Method for Recovery and Immunoaffinity Enrichment of Membrane Proteins Illustrated with Metastatic Ovarian Cancer Tissues

doi: 10.1155/2012/838630

Figure Lengend Snippet: No significant effect of the TD2 buffer was seen in either the rate of color development from the HRP conjugate or antigen affinity in commercial ELISA kits for (a) total human transferrin (Tf), (b and c) total human matrix metalloprotease 2 (MMP2) or 3 (MMP3), (d) total human V-akt murine thymoma viral oncogene homolog 1 (AKT1), or (e) soluble vascular endothelial growth factor receptor (sVEGFR). Apparent affinity constants (±one standard deviation) for separate serial dilutions for the kit standards are shown for each ELISA for both the recommended kit diluent and TD2 buffers. (Standard deviations are determined by partitioning the error of the estimate across the fitted parameters using the Jacobian matrix. In ELISA assays lacking experimental data defining the upper asymptotic limits of quantitation (e.g., VEGF R2 assay of ), this method produces very large errors when fitting the data with a nonlinear equation ).

Article Snippet: The effect of the diluted TD2 buffer on immunoassays was evaluated in several commercial ELISA kits, including human transferrin kit (Bethyl Laboratories, Montgomery, TX), Quantikine MMP-2 and MMP-3 (R&D Systems, Minneapolis, MN), and PathScan total p53 and PathScan total AKT1 (Cell Signaling, Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitation Assay

Journal: International Journal of Proteomics

Article Title: Method for Recovery and Immunoaffinity Enrichment of Membrane Proteins Illustrated with Metastatic Ovarian Cancer Tissues

doi: 10.1155/2012/838630

Figure Lengend Snippet: The concentrations of various protein biomarkers extracted from mixed metastatic ovarian tumors obtained from the omenta of various patients during surgical debulking as measured by ELISA. Proteins were extracted from the cryogenically ground tumor samples using a Barocycler with TD2 Buffer. The concentrations (± one standard deviation) of each biomarker are determined from the extract contained using the ELISA assays described in Figure 3 and extrapolated to that present in the tumor assuming 100% recovery. Transferrin was used as a ubiquitous control protein, which is indicative of the serum content of the sample. Some biomarkers were below the detection limits of the assay. MMP3 was below the limits of quantitation of the ELISA.

Article Snippet: The effect of the diluted TD2 buffer on immunoassays was evaluated in several commercial ELISA kits, including human transferrin kit (Bethyl Laboratories, Montgomery, TX), Quantikine MMP-2 and MMP-3 (R&D Systems, Minneapolis, MN), and PathScan total p53 and PathScan total AKT1 (Cell Signaling, Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Biomarker Assay, Quantitation Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Overexpression of MyrAkt1 in Endothelial Cells Leads to Erythropoietin- and BMP4-Independent Splenic Erythropoiesis in Mice

doi: 10.1371/journal.pone.0055095

Figure Lengend Snippet: Addition of BMP4 to methylcellulose cultures did not increase the number of colonies compared with erythropoietin alone ( A ). Levels of BMP4 mRNA ( B ) and protein ( C ) were unchanged in the spleen. Splenic BMP4+ macrophages recruited in response to chemotherapeutically-induced anemia ( D ) were not found in spleens of control ( E ) or myrAkt1 ( F ) animals (40x). Plasma erythropoietin protein was not significantly elevated until 4 weeks off tetracycline ( E ) p<0.0005, and levels of erythropoietin mRNA were not elevated until 3 weeks off tetracycline in the kidney ( F ) or liver ( G ) p<0.05. Endothelial cells isolated from myrAkt1 kidneys displayed downregulation of phosphorylated and total Akt1 in the presence of tetracycline ( J ). TER119 + erythroblasts showed no significant decrease in Annexin V positivity in spleen or bone marrow ( K ), but TUNEL staining showed a transient decrease in apoptotic nuclei in myrAkt1 spleens at one week p<0.006 ( L) .

Article Snippet: Protein levels were assessed by Western blot with antibodies against phospho-Akt1 (Ser473) (9271, Cell Signaling Technology, Danvers, MA), total Akt1 (9272, Cell Signaling Technology), and GAPDH (374, Millipore, Billerica, MA).

Techniques: Isolation, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: Role of Dystrophin in Airway Smooth Muscle Phenotype, Contraction and Lung Function

doi: 10.1371/journal.pone.0102737

Figure Lengend Snippet: For all panels, day 0 represents protein lysates obtained from serum-fed confluent cultures, and day 7 represents protein lysates obtained from confluent primary tracheal smooth muscle cell cultures (obtained from GR and GRMD animals) after 7-day serum deprivation, with medium changed every 48 h. A: representative western blots for PI3K-GSK3-mTOR signaling pathway proteins. B: densitometry analysis of the effects of serum deprivation on p-Akt1 ( B ), p-GSK3β ( C ) and p-mTOR ( D ) in GR and GRMD tracheal smooth muscle cells are shown. For all histograms β-actin was used as a loading control and phospho-proteins were normalized relative to their respective total protein. Data shown represent means ± SE from 6–9 experiments using 3 different primary tracheal smooth muscle cells obtained from healthy (GR) and dystrophic (GRMD) animals. Statistical comparisons shown were performed by 1-way ANOVA with Tukey’s multiple comparison tests. * p <0.05, ** p <0.01, for GR day 7 versus GRMD day 7.

Article Snippet: Rabbit monoclonal anti-phospho Akt1 (Thr 308), rabbit polyclonal anti-phospho-(Ser9/21)-GSK-3 antibody, rabbit anti-phospho-mTOR (Ser2448), total anti-Akt1, GSK3 and mTOR (Cell Signaling Technology, Beverly, MA, USA, 1 in 1000).

Techniques: Western Blot

Journal: Molecular Cancer

Article Title: BMI1 and Mel-18 oppositely regulate carcinogenesis and progression of gastric cancer

doi: 10.1186/1476-4598-9-40

Figure Lengend Snippet: Mel-18 and BMI1 expression inversely correlates in gastric cancer cell lines and gastric tumors . A) Mel-18 (left) and BMI1 (right) expression in an immortalized human normal gastric epithelial cell line GES-1 and various gastric cancer cell lines as detected by QRT-PCR analysis. The bars indicate mean ± SD from three different experiments. B) BMI1, Mel-18, phospho-AKT (pAKT), total AKT (tAKT), and p16 proteins expression in an immortalized human normal gastric epithelial cell line GES-1 and various gastric cancer cell lines as detected by Western blot analysis. β-actin was used as a loading control. C ) A representative figure of two gastric tumor samples. Sample 1 expresses high Mel-18, low BMI1, low pAKT, and high p16, while the second sample expresses low Mel-18, high BMI1, high pAKT, and low p16. Tissue sections were stained with BMI1, Mel-18-, pAKT-, or p16- specific antibodies and counterstained with hematoxylin as described in experimental procedures.

Article Snippet: Antibodies against human total AKT (tAKT) and phospho-AKT (ser-473, pAKT) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Molecular Cancer

Article Title: BMI1 and Mel-18 oppositely regulate carcinogenesis and progression of gastric cancer

doi: 10.1186/1476-4598-9-40

Figure Lengend Snippet: Reduction of transformed phenotype by Mel-18 overexpression and knockdown of BMI1 expression . A) Overexpression of Mel-18 in SGC-7901 cells results in increased cellular senescence (p < 0.05; upper panel: left, pictures of SA-β-gal stained cells; right, senescent cells were counted and plotted), decreased number of colonies in soft agar (p < 0.01, mid panel: left, pictures of colonies in soft agar, right, the number of colony were counted and plotted); and no difference in apoptosis (lower panel). The bars indicate mean ± SD. Vector infected cells served as a control. B) Knockdown of BMI1 in SGC-7901 results in increased cellular senescence (p < 0.05; upper panel: left, pictures of SA-β-gal stained cells, right, senescent cells were counted and plotted); decreased number of colonies in soft agar (p < 0.05; mid panel: left, pictures of colonies in soft agar, right, the number of colony were counted and plotted); and no difference in apoptosis (lower panel). Ctrli (non-specific sequence) served as control for BMI1 knockdown (BMI1i). C) Overexpression of Mel-18 leads to downregulation of BMI1, and reduction in pAKT (phospho-AKT) and upregulation of p16 expression as determined by Western blot analysis ( left, Mel-18, BMI1, pAKT, tAKT (total AKT), p16, and β-actin were detected by Western blot; right, quantification of the levels of BMI1 and p16, and AKT activity by densitometric analysis as described in Figure 3 ). D) BMI1 knockdown by RNAi approach leads to no change of Mel-18, reduction in pAKT and upregualtion of p16 expression as determined by Western blot analysis (left, pictures of Western blot; right, quantification of the levels of Mel-18 and p16, and AKT activity by densitometric analysis).

Article Snippet: Antibodies against human total AKT (tAKT) and phospho-AKT (ser-473, pAKT) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Transformation Assay, Over Expression, Expressing, Staining, Plasmid Preparation, Infection, Sequencing, Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression

doi: 10.3390/ijms232214283

Figure Lengend Snippet: TKS5 is involved in AKT pathway activation in Y235T, RT4 and UMUC3 cells. ( A ) Protein levels of phospho-AKT (Ser473), phospho-AKT (Thr308) and total-AKT in Y235T parental, Y235T-shScramble and Y235T-shTKS5 cells. ( B ) Protein levels of phospho-AKT (Ser473), phospho-AKT (Thr308) and total-AKT in RT4 parental, RT4-shScramble and RT4-shTKS5 cells. ( C ) Protein levels of phospho-AKT (Ser473), phospho-AKT (Thr308) and total-AKT in UMUC3 parental, UMUC3 empty vector (EV) or UMUC3 with ectopic TKS5 cells. We used the same lysates and membranes to detect the target proteins (TKS5 and AKT) and internal reference protein (β-actin). Please note that one ß-actin membrane served as the loading control for both pAKT targets. We repeated all the experiments three times with different lysates. The quantitation of the blots are presented in the supplemental data .

Article Snippet: We purchased the anti-total-AKT (#75692S) (Western blot dilution: 1:1000), anti-phospho-AKT (Ser473) rabbit polyclonal antibodies (#4060S) (Western blot dilution: 1:1000) and anti-phospho-AKT (Thr308) rabbit polyclonal antibodies (#13038S) (Western blot dilution: 1:1000) from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Plasmid Preparation, Quantitation Assay