Journal: Chemistry & biology

Article Title: Selecting agonists from single cells infected with combinatorial antibody libraries.

doi: 10.1016/j.chembiol.2013.04.012

Figure Lengend Snippet: Figure 5. Signal Transduction by Agonist Antibodies JAK2 was purified from cell lysates using affinity to anti-JAK2 agarose and its phosphorylation was detected with western blotting using anti- phosphotyrosine antibodies. Phosphorylation of STAT3, STAT5, Akt, and MAPK induced by agonist antibodies or rhTPO stimulation was detected with direct western blotting of cell lysates. See also Figure S6.

Article Snippet: The immunoprecipitates were analyzed with western blotting using anti-phospho-JAK2 (Tyr1007/1008) (C80C3) antibody (Cell Signaling Technology, catalog no. 3776) and anti-total JAK2 antibody (C-14) (Santa Cruz Biotechnology, catalog no. sc-34479).

Techniques: Transduction, Phospho-proteomics, Western Blot

Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Evidence of Abelson helper integration site 1 (Ahi-1)–Janus kinase 2 (Jak2) and Ahi-1-BCR-ABL interactions in BCR-ABL – and Jak2 -transduced cells coexpressing Ahi-1 and its mutants. A) Schematic of functional domains of full-length and mutant (N-ter∆) Ahi-1. Vectors were transduced into BaF3 cells and BCR-ABL –inducible cells, and increased protein expression levels of full-length and mutant Ahi-1 were detected by Western blotting. B) Ahi-1 ( left panel ) or Jak2 ( right panel ) were immunoprecipitated from lysates of the same transduced cells and then electrophoresed and probed with specific antibodies, as indicated. C) Human influenza hemagglutinin (HA)–tagged SH3 ∆ or SH3WD40 ∆ mutants were transfected into 293T cells with or without a Jak2 vector, immunoprecipitated with antibodies to either HA or Jak2, electrophoresed, and probed using Jak2 or Ahi-1 antibodies ( left panel ). The right panel shows that these two mutants were coexpressed with BCR-ABL , immunoprecipitated with an anti-HA antibody, and probed with a c-ABL antibody. A schematic of the functional domains of full-length Ahi-1 and its SH3 ∆ and SH3WD40 ∆ mutants is below the right panel. IP = immunoprecipitation; WB = Western blotting.

Article Snippet: The antibodies in this study included a rabbit polyclonal N-terminal AHI-1 antibody and an antimouse Ahi-1 mouse monoclonal antibody (1:1000 dilution) (C-mAhi-1 M5, Applied Biological Materials Inc Vancouver, BC, Canada), an antimouse ABL mouse monoclonal antibody (1:1000 dilution) (8E9; BD Biosciences), an antimouse JAK2 rabbit monoclonal antibody (1:1000 dilution) (Cell signaling Technology, MA), a rabbit polyclonal anti-JAK2 agarose conjugated antibody (Santa Cruz Biotechnology), anti-phospho-JAK2 rabbit polyclonal antibodies (1:1000 dilution) (Cell Signaling and Epitomics Inc, Burlingame, CA), an antihuman STAT5 rabbit polyclonal antibody (1:1000 dilution) (Millipore, Billeria, MA), an anti-phospho-STAT5 rabbit monoclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-CRKL rabbit polyclonal antibody (1:2000 dilution) (Cell Signaling), an antihuman CRKL rabbit polyclonal antibody (1:1000 dilution) (Santa Cruz Biotechonolgy), an antiphosphotyrosine mouse monoclonal antibody (4G10) (1:2000 dilution) (Millipore, Billerica, MA), an anti-phospho-AKT rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antimouse Akt rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-ERK rabbit polyclonal antibody (1:1000 dilution), an antirat ERK rabbit polyclonal (1:1000 dilution), an antimouse GAPDH mouse monoclonal antibody (1:2000 dilution) (Sigma Aldrich), an antiactin mouse monoclonal antibody (1:1000 dilution) (Sigma Aldrich), and an anti-HA mouse monoclonal antibody (1:1000 dilution) (Applied Biological Materials Inc).

Techniques: Functional Assay, Mutagenesis, Expressing, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Effect of imatinib (IM) on induction of apoptosis and inhibition of colony formation in BCR-ABL –transduced cells coexpressing Abelson helper integration site 1 ( Ahi-1 ) mutants and combined treatment with IM and a Janus kinase 2 (JAK2) inhibitor on inhibition of growth of AHI-1 –overexpressing and IM-resistant cells. A) BCR-ABL –transduced cells coexpressing full-length Ahi-1 and its mutants were cultured with IM, and the treated cells were stained with Annexin V/7-aminoactinomysin (7-AAD) to detect apoptotic cells after 24 or 48 hours. Representative fluorescence-activated cell sorting plots showing detection of Annexin V/7-AAD + cells 24 hours after IM treatment are included. B) Transduced cells (200 cells/group) were pretreated with IM for 24 hours and then plated in colony-forming cell assays. Colony numbers produced in semisolid media were counted and expressed as a percentage of counts obtained from control cells without any drug added. C–E) Control K562 cells, AHI-1 –transduced SH4-bulk K562 cells (with suppressed AHI-1 expression), lenti-AHI-1 K652 cells (overexpressing AHI-1 ) and IM-resistant K562 cells were incubated with IM ( C ), or TG101209 (TG) alone ( D ), or both in combination ( E ), or with no drug for 48 hours and the percentage of viable cells was then measured. F) AHI-1 was immunoprecipitated from cell lysates of AHI-1 –overexpressing K562 cells treated with IM and TG alone or in combination for 16 hours. The immunoprecipitates were then probed with an antityrosine phosphorylation antibody (4G10), a BCR-ABL antibody, a JAK2 antibody, and an AHI-1 antibody. Control cells = Hut78 cells. Data are means, and error bars represent 95% confidence intervals from three independent experiments in triplicate. P values were calculated using a two-sided Student t test. WB = Western blotting.

Article Snippet: The antibodies in this study included a rabbit polyclonal N-terminal AHI-1 antibody and an antimouse Ahi-1 mouse monoclonal antibody (1:1000 dilution) (C-mAhi-1 M5, Applied Biological Materials Inc Vancouver, BC, Canada), an antimouse ABL mouse monoclonal antibody (1:1000 dilution) (8E9; BD Biosciences), an antimouse JAK2 rabbit monoclonal antibody (1:1000 dilution) (Cell signaling Technology, MA), a rabbit polyclonal anti-JAK2 agarose conjugated antibody (Santa Cruz Biotechnology), anti-phospho-JAK2 rabbit polyclonal antibodies (1:1000 dilution) (Cell Signaling and Epitomics Inc, Burlingame, CA), an antihuman STAT5 rabbit polyclonal antibody (1:1000 dilution) (Millipore, Billeria, MA), an anti-phospho-STAT5 rabbit monoclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-CRKL rabbit polyclonal antibody (1:2000 dilution) (Cell Signaling), an antihuman CRKL rabbit polyclonal antibody (1:1000 dilution) (Santa Cruz Biotechonolgy), an antiphosphotyrosine mouse monoclonal antibody (4G10) (1:2000 dilution) (Millipore, Billerica, MA), an anti-phospho-AKT rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antimouse Akt rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-ERK rabbit polyclonal antibody (1:1000 dilution), an antirat ERK rabbit polyclonal (1:1000 dilution), an antimouse GAPDH mouse monoclonal antibody (1:2000 dilution) (Sigma Aldrich), an antiactin mouse monoclonal antibody (1:1000 dilution) (Sigma Aldrich), and an anti-HA mouse monoclonal antibody (1:1000 dilution) (Applied Biological Materials Inc).

Techniques: Inhibition, Cell Culture, Staining, Fluorescence, FACS, Produced, Control, Expressing, Incubation, Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Inhibition of phosphorylation of BCR-ABL, CRK-like (CRKL), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 5 (STAT5) in transduced K562 cells in response to combined treatment with TG101209 (TG) and imatinib (IM). A) K562 cells, Abelson helper integration site 1 ( AHI-1 )–overexpressing K562 cells, and IM-resistant K562 cells (IMR) were cultured with or without IM, TG, or a combination of IM and TG for 16 hours. Western blot analysis detected phosphorylation and protein expression of various proteins using the specific antibodies indicated. B) BV173 cells were treated with IM, TG, or IM plus TG for 16 hours. Cell lysates were immuno-probed with the specific antibodies indicated. GAPDH or actin was utilized as a loading control.

Article Snippet: The antibodies in this study included a rabbit polyclonal N-terminal AHI-1 antibody and an antimouse Ahi-1 mouse monoclonal antibody (1:1000 dilution) (C-mAhi-1 M5, Applied Biological Materials Inc Vancouver, BC, Canada), an antimouse ABL mouse monoclonal antibody (1:1000 dilution) (8E9; BD Biosciences), an antimouse JAK2 rabbit monoclonal antibody (1:1000 dilution) (Cell signaling Technology, MA), a rabbit polyclonal anti-JAK2 agarose conjugated antibody (Santa Cruz Biotechnology), anti-phospho-JAK2 rabbit polyclonal antibodies (1:1000 dilution) (Cell Signaling and Epitomics Inc, Burlingame, CA), an antihuman STAT5 rabbit polyclonal antibody (1:1000 dilution) (Millipore, Billeria, MA), an anti-phospho-STAT5 rabbit monoclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-CRKL rabbit polyclonal antibody (1:2000 dilution) (Cell Signaling), an antihuman CRKL rabbit polyclonal antibody (1:1000 dilution) (Santa Cruz Biotechonolgy), an antiphosphotyrosine mouse monoclonal antibody (4G10) (1:2000 dilution) (Millipore, Billerica, MA), an anti-phospho-AKT rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antimouse Akt rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-ERK rabbit polyclonal antibody (1:1000 dilution), an antirat ERK rabbit polyclonal (1:1000 dilution), an antimouse GAPDH mouse monoclonal antibody (1:2000 dilution) (Sigma Aldrich), an antiactin mouse monoclonal antibody (1:1000 dilution) (Sigma Aldrich), and an anti-HA mouse monoclonal antibody (1:1000 dilution) (Applied Biological Materials Inc).

Techniques: Inhibition, Phospho-proteomics, Cell Culture, Western Blot, Expressing, Control

Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Effects of oral administration of TG101209 (TG) and imatinib (IM) on elimination of chronic myeloid leukemia (CML) BV173 cells and survival of leukemic mice in immunodeficient mice. A) BV173 cells were cultured with 1.0 µM IM, 0.5 µM TG, IM plus TG, or no drug for 3 days, and the progeny recovered from 2.5×10 6 initial cells were then injected intravenously into NOD/SCID–interleukin 2 receptor γ–chain-deficient (NSG) mice (n = 6 mice per condition). The left panel shows fluorescence-activated cell sorting profiles of engrafted human CD19/20 + cells detected in bone marrow aspirates of representative mice obtained 3 weeks posttransplant. The percentage of human CD19/20 + BV173 cells detected in the bone marrow of mice examined 3 weeks after injection is shown in the right panel . Data are means, and error bars represent 95% confidence intervals of six measurements per condition. B) Survival curve for recipients of BV173 cells (2.5×10 6 per mouse; n = 6 mice per group) treated by oral gavage beginning at 2 weeks posttransplant with vehicle, IM (50mg/kg), TG (60mg/kg), and IM (50mg/kg) plus TG (60mg/kg) twice a day for 2 weeks. Statistically significantly prolonged survival was observed in mice receiving the combination treatment ( left panel ). Body weights of mice in each treated group were measured, as indicated in the right panel . Log-rank tests were used to compare median survival of different groups (n = 6 mice per group), and P values were calculated using a two-sided Student t test. C) Model of the mechanism by which Abelson helper integration site 1 (AHI-1)–BCR-ABL and AHI-1–Janus kinase 2 (JAK2) interactions regulate constitutive activation of BCR-ABL and JAK2/ signal transducer and activator of transcription 5 (STAT5), resulting in increased leukemic stem cell proliferation, survival and maintenance and reduced tyrosine kinase inhibitor (TKI) response of these cells. Targeting both BCR-ABL and JAK2 activities to destabilize this complex perturbs these biological properties. IL-3 = interleukin 3; IL-3R = interleukin 3 receptor.

Article Snippet: The antibodies in this study included a rabbit polyclonal N-terminal AHI-1 antibody and an antimouse Ahi-1 mouse monoclonal antibody (1:1000 dilution) (C-mAhi-1 M5, Applied Biological Materials Inc Vancouver, BC, Canada), an antimouse ABL mouse monoclonal antibody (1:1000 dilution) (8E9; BD Biosciences), an antimouse JAK2 rabbit monoclonal antibody (1:1000 dilution) (Cell signaling Technology, MA), a rabbit polyclonal anti-JAK2 agarose conjugated antibody (Santa Cruz Biotechnology), anti-phospho-JAK2 rabbit polyclonal antibodies (1:1000 dilution) (Cell Signaling and Epitomics Inc, Burlingame, CA), an antihuman STAT5 rabbit polyclonal antibody (1:1000 dilution) (Millipore, Billeria, MA), an anti-phospho-STAT5 rabbit monoclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-CRKL rabbit polyclonal antibody (1:2000 dilution) (Cell Signaling), an antihuman CRKL rabbit polyclonal antibody (1:1000 dilution) (Santa Cruz Biotechonolgy), an antiphosphotyrosine mouse monoclonal antibody (4G10) (1:2000 dilution) (Millipore, Billerica, MA), an anti-phospho-AKT rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antimouse Akt rabbit polyclonal antibody (1:1000 dilution) (Cell Signaling), an antihuman phospho-ERK rabbit polyclonal antibody (1:1000 dilution), an antirat ERK rabbit polyclonal (1:1000 dilution), an antimouse GAPDH mouse monoclonal antibody (1:2000 dilution) (Sigma Aldrich), an antiactin mouse monoclonal antibody (1:1000 dilution) (Sigma Aldrich), and an anti-HA mouse monoclonal antibody (1:1000 dilution) (Applied Biological Materials Inc).

Techniques: Cell Culture, Injection, Fluorescence, FACS, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

doi: 10.1073/pnas.1700682114

Figure Lengend Snippet: JAK1 (and JAK2) are required for cell survival in JAK inhibitor-sensitive cells. (A) shJAK1 induced cell death in all JAK inhibitor-sensitive cells. The percentage of viable shRNA+ cells at days 2, 4, 6, 8, 10, and 12 after addition of doxycycline was compared with that of day 0. shRNA-infected cells were GFP+; the shRNA expression was induced by doxycycline. shSC4 served as a negative control; shRPL6, as a positive control. (B) shJAK2 induced cell death in Mac-1/2A/2B cells. (C) Western blot analysis of JAK1 and p-STAT3 in Mac-1 cells stably infected with retroviral shJAK1. The shRNA expression was induced by doxycycline for 24 h or 72 h. (D) Western blot analysis of PCM1-JAK2 and p-STAT3 in Mac-1 cells stably infected with retroviral shJAK2.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology: JAK1, JAK2 (3230), STAT3, and p-STAT3 (9145).

Techniques: shRNA, Infection, Expressing, Negative Control, Positive Control, Western Blot, Stable Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

doi: 10.1073/pnas.1700682114

Figure Lengend Snippet: JAK and STAT3 mutations in JAK inhibitor-sensitive cells

Article Snippet: The following antibodies were purchased from Cell Signaling Technology: JAK1, JAK2 (3230), STAT3, and p-STAT3 (9145).

Techniques: Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

doi: 10.1073/pnas.1700682114

Figure Lengend Snippet: Western blot analysis of JAK2 and PCM1-JAK2.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology: JAK1, JAK2 (3230), STAT3, and p-STAT3 (9145).

Techniques: Western Blot

Journal: Endocrinology

Article Title: Insulin-Like Growth Factor-Independent Effects of Growth Hormone on Growth Plate Chondrogenesis and Longitudinal Bone Growth.

doi: 10.1210/en.2014-1983

Figure Lengend Snippet: Figure 6. Effects of systemic GH and IGF-1 on JAK2, STAT5B, and Akt in the tibial growth plate of KO and C mice. Three- week old KO and C mice (n 4–7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. At the end of the treatment period, protein extracted from the tibial growth plates was electrophoresed and immunoblotted with antibodies directed to phosphorylated and total proteins. A representative blot from three independent experiments is presented for each protein.

Article Snippet: Chondrocytes were harvested and immunoblotting was performed using equal amounts of protein (50–100 g) and the following primary antibodies: anti- actin (Cat. No.A-2066; Sigma), p-NF- B p65 (SC-33 039, Santa Cruz), rabbit polyclonal antibody against NF- B p65 (SC-372,Santa Cruz), p-Jak2 (Cat#9356, Cell Signaling Technology Inc., Danvers, MA), rabbit polyclonal antibody against Jak2 (SC-294, Santa Cruz), rabbit polyclonal antibody against p-STAT5 (Cat#4322, Cell Signaling Technology Inc.),rabbitpolyclonal antibody against STAT5 (SC-835,SantaCruz), goat polyclonal antibody against BMP-2 (SC-6895, Santa Cruz).

Techniques:

Journal: Oncology reports

Article Title: Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

doi: 10.3892/or.2017.5588

Figure Lengend Snippet: Figure 2. Silibinin downregulates the expression of Jak2/STAT3 signaling proteins in a dose- and time-dependent manner. (A) Western blot analyses showing the concentration dependent effect of silibinin in MDA‑MB‑231 cells following exposure to silibinin for 24 h. (B) Relative levels of the pSTAT3, STAT3, pJak2, and Jak2 proteins. (C) Time-dependent effect of silibinin on protein expression in MDA‑MB‑231 cells. (D) Relative expression levels of pSTAT3, STAT3, pJak2, and Jak2, measured using densitometry. These data were normalized to actin levels, and then shown as a percentage of the control. The data presented are representative of three independent experiments. Statistical analyses were conducted using the t-test (**p<0.01, ***p<0.001).

Article Snippet: An anti phosphor Jak2 antibody (Tyrosine residue 1007/1008) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Control

Journal: Oncology reports

Article Title: Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

doi: 10.3892/or.2017.5588

Figure Lengend Snippet: Figure 7. Schematic representation of the inhibition of invasive mechanisms provoked by silibinin in MDA‑MB‑231 cells. Silibinin inhibits Jak2 expression and phosphorylation, resulting, in turn, in the inhibition of STAT3 expression, phosphorylation, nuclear translocation, and DNA binding activity. Consequently, STAT3's down-stream targets are inhibited (including MMP2), resulting in reduced cell migration and invasion.

Article Snippet: An anti phosphor Jak2 antibody (Tyrosine residue 1007/1008) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing, Phospho-proteomics, Translocation Assay, Binding Assay, Activity Assay, Migration

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay