rabbit polyclonal anti human phosphorylated stat5  (Bioss)

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Silencing of MRCKα inhibits the stimulatory effects of Met and Leu on mRNA abundance of genes related to milk protein synthesis in bovine mammary epithelial cells. Cells were transfected with scrambled siRNA (control, SC) or MRCKα siRNA (SI) and stimulated simultaneously with control (B, basal medium), additional Met (M, Met = 0.6 mM) or Leu (L, Leu = 0.6 mM). After 24 h, cells were collected and isolated for RT-qPCR analysis. Values are presented as mean ± SEM for three independent experiments. P AA , P -value of amino acid addition (AA); P SI , P -value of silencing of MRCKα (SI); P AA×SI , P -value of AA × SI interaction. CSN2 = β-casein; CSN1S1 = αS1-casein; CSN3 = κ-casein; LALBA = lactalbumin α; MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; mTOR = mechanistic target of rapamycin; JAK2 = Janus kinase 2; STAT5 = signal transducers and activators of transcription 5.

Article Snippet: Rabbit polyclonal anti-human signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

Techniques: Transfection, Control, Isolation, Quantitative RT-PCR, Binding Assay

Journal: Oncogene

Article Title: Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

doi: 10.1038/sj.onc.1206028

Figure Lengend Snippet: Figure 1 PD180970 inhibits Bcr – Abl tyrosine phosphorylation and Stat5 activation in the Ph+ CML cell line K562. (a) Immu- noprecipitations were performed with anti-Abl antibody using ly- sates prepared from K562 cells treated for 12 h with DMSO vehicle (lane 1) or increasing concentrations of PD180970 (lanes 2 – 5) and subjected to Western blot analyses. Upper panel shows the levels of Abl tyrosine phosphorylation as detected by anti- phosphotyrosine (anti-pTyr) antibody. The lower panel was probed with anti-Abl antibody to confirm equal loading of pro- teins. Both Bcr – Abl (210 kd) and c-Abl (145 kd) proteins are de- tected by these antibodies. IP, immunoprecipitation; IB, immunoblot. (b) EMSA was performed to measure levels of Stat5 DNA-binding activity using nuclear extracts prepared from K562, HEL92.1.7 or HL-60 cells treated with the indicated doses of PD180970 for 24 h. The 32P-labeled MGFe probe was used that specifically binds Stat5 in this assay. The position of activated Stat5 bound to probe in the gel is indicated. (c) EMSA was per- formed to determine the kinetics of Stat5 inhibition following treatment with PD180970 in K562 cells for the indicated times, with redosing at 12 h for the 24 h time point. Experiments were repeated at least three times with similar results. Control (Con) lanes were treated with DMSO vehicle alone

Article Snippet: Immunocytochemistry to detect activated phospho-Stat5 protein Immunostaining for phospho-Stat5 was performed using a rabbit anti-human polyclonal antibody against tyrosine phosphorylated Stat5 (Phospho-Stat5 Tyr694; Cell Signaling, Beverly, MA, USA).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Immunoprecipitation, Binding Assay, Activity Assay, Labeling, Inhibition, Control

Journal: Oncogene

Article Title: Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

doi: 10.1038/sj.onc.1206028

Figure Lengend Snippet: Figure 4 Expression of dominant-negative Stat5 protein induces apoptosis of K562 cells. Cells were either mock infected (a) or in- fected with control vaccinia virus (b) or vaccinia virus encoding dominant-negative Stat5 protein (c). Viral infections of K562 cells were carried out as described in Materials and methods. After 24 h of infection, apoptosis was measured by Annexin V-FITC staining followed by flow cytometry

Article Snippet: Immunocytochemistry to detect activated phospho-Stat5 protein Immunostaining for phospho-Stat5 was performed using a rabbit anti-human polyclonal antibody against tyrosine phosphorylated Stat5 (Phospho-Stat5 Tyr694; Cell Signaling, Beverly, MA, USA).

Techniques: Expressing, Dominant Negative Mutation, Infection, Control, Virus, Staining, Cytometry

Journal: Oncogene

Article Title: Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

doi: 10.1038/sj.onc.1206028

Figure Lengend Snippet: Figure 6 Inhibition of Stat5 DNA-binding activity in primary CML patient specimens treated ex vivo with PD180970. Fresh peripheral blood CD34+ mononuclear cells purified as described in Materials and methods were incubated ex vivo with PD180970 and then assayed for levels of Stat5 DNA-binding ac- tivity and apoptosis. (a) EMSA of Stat5 DNA binding to radiola- beled MGFe probe after 24 h treatment with PD180970 at the indicated concentrations or DMSO (Con) vehicle alone. The po- sitive control (Pos Con) was K562 cells, and the star indicates the position of band that was supershifted by anti-Stat5 (lane 10) antibodies. (b) Apoptosis was determined after 48 h treatment with DMSO or 250 nM PD180970 for one patient specimen. Staining with 7-AAD (vertical axis) is indicative of total dead cells, while Annexin-V staining (horizontal axis) is specific for apoptotic cells. Percentages indicate the apoptosis-specific cell death. Similar results were obtained with a second CML patient specimen (patient no. 5, data not shown)

Article Snippet: Immunocytochemistry to detect activated phospho-Stat5 protein Immunostaining for phospho-Stat5 was performed using a rabbit anti-human polyclonal antibody against tyrosine phosphorylated Stat5 (Phospho-Stat5 Tyr694; Cell Signaling, Beverly, MA, USA).

Techniques: Inhibition, Binding Assay, Activity Assay, Ex Vivo, Incubation, Control, Staining

Journal: Oncogene

Article Title: Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

doi: 10.1038/sj.onc.1206028

Figure Lengend Snippet: Figure 7 Immunocytochemical detection of activated phospho-Stat5 in K562 cells and primary leukemic blasts after treatment ex vivo with PD180970. Cytospins of K562 cells or purified CML patient CD34+ mononuclear cells were immunostained with anti- bodies to phosphotyrosine-Stat5 as described in Materials and methods. Cells were incubated ex vivo for 24 h with PD180970 prior to immunostaining. (A1 – C1) K562 cells were treated with DMSO vehicle alone or the indicated concentrations of PD180970. (A2 – C2) CD34+ mononuclear cells were purified from the peripheral blood of a CML patient (patient no. 5) and treated with DMSO or the indicated concentrations of PD180970 ex vivo

Article Snippet: Immunocytochemistry to detect activated phospho-Stat5 protein Immunostaining for phospho-Stat5 was performed using a rabbit anti-human polyclonal antibody against tyrosine phosphorylated Stat5 (Phospho-Stat5 Tyr694; Cell Signaling, Beverly, MA, USA).

Techniques: Ex Vivo, Incubation, Immunostaining

Journal: Oncogene

Article Title: Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

doi: 10.1038/sj.onc.1206028

Figure Lengend Snippet: Figure 8 PD180970 inhibits Stat5 signaling and induces apoptosis in Bcl – Abl-positive leukemic cells resistant to STI-571. (a) EMSA was performed to measure levels of Stat5 DNA-binding activities in nuclear extracts prepared from K562 cells and HL- 60BA cells expressing ectopic Bcl – Abl protein, as well as their STI-571 resistant counterparts, Re-K562 and Re-HL-60BA. The Re-K562 cells have lost Bcl – Abl expression, whereas Re-HL-60BA cells have retained Bcl – Abl expression. Cells were treated with DMSO vehicle or 50 nM PD180970 as indicated for 24 h. (b) Cell lines in a were treated with DMSO or 50 nM PD180970 for 48 h and then assayed for apoptosis by flow cytometry using combined Annexin-V and 7-AAD staining

Article Snippet: Immunocytochemistry to detect activated phospho-Stat5 protein Immunostaining for phospho-Stat5 was performed using a rabbit anti-human polyclonal antibody against tyrosine phosphorylated Stat5 (Phospho-Stat5 Tyr694; Cell Signaling, Beverly, MA, USA).

Techniques: Binding Assay, Expressing, Cytometry, Staining