Journal: Developmental biology

Article Title: Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development.

doi: 10.1016/j.ydbio.2011.08.027

Figure Lengend Snippet: Fig. 2. Generation of mstnC315Y medaka. (A) Sequence data for each genotype. Lines in green, red, black and blue indicate adenine, thymine, guanine and cytosine, respectively. Three-letter amino-acid codes show the resulting transcript. Top figure illustrates WT medaka sequence, while middle and bottom figures illustrate heterozygous and homozygous C315Y mutation, respectively. Guanine to adenine nucleotide transition causes an amino-acid change from Cys to Tyr at amino acid 315 (black triangles). (B) Demonstration of de- ficient medaka for the MSTN signaling pathway. Phosphorylated Smad2 in nuclear proteins were targeted and detected with western blotting. Nucleus protein were obtained from muscles of WT (left lane) and mstnC315Y (middle lane) at 16 wk post-hatching. Upper figure shows decreased expression of phosphorylated Smad2 in mstnC315Y. TGF-β treated Mv1Lu cell lysates were used as positive control of phosphorylated Smad2 for western blotting (right lane). Lower figure shows histone H2B used as a loading control.

Article Snippet: TGF-β treated Mv1Lu cell lysates (Santa Cruz Biotechnology, Inc., CA, USA) were used as a positive control for phosphorylated Smad2 protein.

Techniques: Sequencing, Mutagenesis, Western Blot, Muscles, Expressing, Positive Control, Control

Journal: The Journal of biological chemistry

Article Title: c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells.

doi: 10.1074/jbc.273.50.33714

Figure Lengend Snippet: FIG. 1. c-Src kinase activity is elevated in SP1 carcinoma cells compared with Mv1Lu epithelial cells. Cell lysates were prepared from serum-starved Mv1Lu and SP1 cells treated without (2) or with (1) HGF (40 ng/ml) for 10 min and were immunoprecipitated with anti-c-Src IgG. Immunoprecipitates were subjected to an in vitro kinase assay using enolase as a substrate, and kinase activity was measured as described under “Experimental Procedures.” A, autoradiogram show- ing 32P-labeled enolase. B, quantitation of autoradiogram using Phos- phorImager. Results are expressed as the percentage of cpm in un- treated Mv1Lu cells (100%), normalized to the amount of c-Src protein in C. The means 6 range of two experiments are shown. Similar results were obtained using the c-Src kinase family-specific cdc2 peptide as substrate (data not shown). C, Western blot analysis of immmunopre- cipitates in A, probed with anti-c-Src IgG.

Article Snippet: Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above.

Techniques: Activity Assay, Immunoprecipitation, In Vitro, Kinase Assay, Labeling, Quantitation Assay, Western Blot

Journal: The Journal of biological chemistry

Article Title: c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells.

doi: 10.1074/jbc.273.50.33714

Figure Lengend Snippet: FIG. 2. c-Src kinase binds to tyrosine-phosphorylated Met. Cell lysates derived from serum-starved Mv1Lu cells treated without (2) or with (1) HGF (40 ng/ml) for 15 min were immunoprecipitated with anti-c-Src IgG (A) or anti-Met IgG (B). The immune complexes were separated by 8% SDS-PAGE and immunoblotted with anti-Met IgG (A) or anti-c-Src IgG (B). Protein molecular mass standards are shown on the right. This experiment was done twice with similar results.

Article Snippet: Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above.

Techniques: Derivative Assay, Immunoprecipitation, SDS Page

Journal: The Journal of biological chemistry

Article Title: c-Src kinase activity is required for hepatocyte growth factor-induced motility and anchorage-independent growth of mammary carcinoma cells.

doi: 10.1074/jbc.273.50.33714

Figure Lengend Snippet: FIG. 5. Expression of dominant negative mutant SRC-RF does not alter Met protein levels or activity and downstream signal- ing. A, SP1 cells transfected with SRC-RF or SRC or untreated SP1 cells were prestarved overnight and lysed as described in the legend to Fig. 1. Equal amounts of protein from each lysate were concentrated on Microcon 10 filters (Amicon Inc., Beverly, MA) and analyzed by Western blotting with anti-Met IgG (top panel). The blot was stripped and reprobed with anti-phosphotyrosine antibody (middle panel). Cell ly- sates were also immunoprecipitated with anti-Met IgG, and immuno- precipitates were subjected to an in vitro Met kinase assay as described under “Experimental Procedures.” The autoradiogram depicting 32P- labeling of Met is shown (bottom panel). Relative band intensities and amount of 32P labeling was determined using a Storm PhosphorImager. The relative amount of Met tyrosine phosphorylation (1.0, 1.0, or 1.0) or of in vitro Met autophosphorylation (1.0, 1.1, or 1.0) was not signifi- cantly different among the three cell lines. B, serum-starved SP1 cells transfected with SRC-RF or SRC and untreated SP1 cells were lysed as described in the legend to Fig. 1. Prestarved Mv1Lu cells untreated or treated with HGF (40 ng/ml) for 10 min were used as negative and positive controls, respectively. Equal amounts of protein from each lysate were immunoprecipitated with anti-PCL-g1 IgG. Immunopre- cipitates were subjected to 7% SDS-PAGE and transferred to nitrocel- lulose. The blot was probed with anti-PCL-g1 IgG (top panel) before being stripped and reprobed with anti-phosphotyrosine antibody (bot- tom panel). This experiment was done twice with similar results. IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above.

Techniques: Expressing, Dominant Negative Mutation, Activity Assay, Transfection, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Labeling, Phospho-proteomics, SDS Page