Journal: Oncogene

Article Title: An activating mutation in the transmembrane domain of the granulocyte colony-stimulating factor receptor in patients with acute myeloid leukemia.

doi: 10.1038/sj.onc.1205767

Figure Lengend Snippet: Figure 5 Tyrosine phosphorylation of (a) anti-JAK2 and (b) anti-G-CSFR immunoprecipitates from Ba/F3 transfectants sti- mulated with or without 100 ng/ml G-CSF for 3 min or 10 ng/ ml mIL-3 for 10 min. Lysates were immunoprecipitated with anti-JAK2 or anti-G-CSFR antibodies; blots were probed with the phospho-tyrosine specific antibody PY99, stripped and re- probed with antibody against JAK2 or G-CSFR

Article Snippet: Western blots were probed with PY99, a phosphotyrosine specific antibody (Santa Cruz Biotechnology), then stripped and re-probed with antibody against JAK2 or the receptor.

Techniques: Phospho-proteomics, Immunoprecipitation

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Hypoxia‐induced JAK2/STAT3 activation in a PAH mouse model and HPASMCs. Representative results for coimmunostaining of p‐JAK2 and α‐SMA (A), p‐STAT3 and α‐SMA (B) in lung sections from WT mice exposed to normoxia or hypoxia (n = 3 per group). Confocal immunofluorescence images for coimmunostaining of p‐JAK2 and p‐STAT3 in HPASMCs following normoxic or hypoxic exposure (C). All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. *** P < .001

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Activation Assay, Immunofluorescence

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Generation of SMC‐specific Jak2 ‐knockout mice. Schematic diagram of transgenic mice used to generate Jak2‐CKO and Jak2‐C mice (A). PCR analysis of tail genomic DNA to determine the presence of the floxed null allele (B). Western blot analysis to confirm Jak2 depletion in pulmonary arteries (C; n = 3 per group) and cardiac muscle (D; n = 3 per group). Immunohistochemistry analysis to confirm Jak2 depletion in PASMCs (E; n = 3 per group). Coimmunostaining results of p‐STAT3 and α‐SMA in lung sections from Jak2‐C and Jak2‐CKO mice (F). All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. *** P < .001

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Knock-Out, Transgenic Assay, Western Blot, Immunohistochemistry

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: SMC‐specific Jak2 deficiency improved the reaction of pulmonary blood vessels to hypoxic condition. RVSP (A), RV/(LV + S) ratio (B), PAT/PT ratio (C), mean systemic arterial BP (D), ejection fraction (E), fractional shortening (F) and body weight change ratio (G) in Jak2‐C and Jak2‐CKO mice after exposure to normoxic (n = 8 per group) or hypoxic (n = 10 per group) conditions for 28 days. The data are represented as the mean ± SEM. ** P < .01. RVSP, right ventricular systolic pressure; RV/ (LV+S), the right ventricle/left ventricle plus septum; PAT/PT, pulmonary acceleration time/pulmonary ejection time

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques:

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Loss of Jak2 in smooth muscle cells protected against pulmonary vascular remodelling after hypoxia. Representative HE‐stained (top) and EVG‐stained (bottom) sections (A), quantification of pulmonary arteriole wall thickness (B) and α‐SMA immunostaining (C) in the lungs of Jak2‐C and Jak2‐CKO mice after normoxic (n = 8 per group) or hypoxic (n = 10 per group) exposure for 28 days. Ten vessels were analysed per mouse. Coimmunostaining results of α‐SMA and Ki67 in lung sections from Jak2‐C and Jak2‐CKO mice after hypoxic (D; n = 10 per group) exposure for 28 days. All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. * P < .05; ** P < .01. HE, haematoxylin and eosin; EVG, elastic van gieson; PA, pulmonary artery

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Staining, Immunostaining

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Hypoxia‐induced HPASMC proliferation was suppressed by a JAK2 inhibitor. Western blot analysis of p‐JAK2, JAK2, p‐STAT3 and STAT3 in HPASMCs (A). CCK‐8 analysis of HPASMCs pre‐treated with different concentrations of TG for 1 h following 24 h hypoxic exposure (B). CFSE dilution analysis (C), EdU staining (D) and cell cycle analysis (E) of HPASMCs pre‐treated with DMSO or TG for 1 h following 24 h hypoxic exposure. All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. * P < .05; ** P < .01; *** P < .001. TG, TG‐101348; CFSE, carboxyfluorescein diacetate succinimidyl ester; EdU, 5‐ethynyl‐uridine; DMSO, dimethyl sulphoxide

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Western Blot, CCK-8 Assay, Staining, Cell Cycle Assay

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: JAK2/STAT3 promoted HPASMC proliferation programming by enhancing cyclin A2 expression following hypoxic exposure. Real‐time PCR to determine cyclin A2 (A), cyclin D1 (B), cyclin E1 (C), CDK2 (D) and CDK4 (E) expression in HPASMCs. Western blot analysis of cyclin A2 expression in HPASMCs (F). ChIP‐PCR results for analysis of p‐STAT3 binding activity to the CNNA2 promoter (G). Results for CNNA2 promoter luciferase reporter assays in HPASMCs (H). Coimmunostaining results of α‐SMA and cyclin A2 in lung sections from Jak2‐C and Jak2‐CKO mice after hypoxic (I; n = 10 per group) exposure for 28 days. All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. * P < .05; ** P < .01; *** P < .001. CDK2, cyclin‐dependent kinase 2; CDK4, cyclin‐dependent kinase 4

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Activity Assay, Luciferase

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Diagram of the mechanisms underlying JAK2 regulation of PAH. Hypoxia induced PASMC proliferation through promoting the JAK2/STAT3/cyclin A2 pathway, in which STAT3 bound directly to the CCNA2 promoter and transcriptionally activated cyclin A2 under hypoxic condition, ultimately leading to increased pulmonary arterial remodelling in PAH

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2017.7561

Figure Lengend Snippet: Primer sequences.

Article Snippet: The membranes were then exposed to the primary antibodies against JAK2 (1:2,000; cat. no. 17670-1-AP; ProteinTech Group, Inc.) and STAT3 (1:1,000; cat. no. 60199-1-lg; ProteinTech Group, Inc) overnight at 4°C. β-actin (1:1,000; cat. no. 60008-1-lg; ProteinTech Group, Inc) was used as an internal control for protein loading.

Techniques: Sequencing, Amplification

Journal: Molecular Medicine Reports

Article Title: Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2017.7561

Figure Lengend Snippet: Effect of mXCHD serum on the mRNA expression levels of JAK2 and STAT3 in HepG2.2.15 cells. Following treatment for 48 h, the cells were collected, and the mRNA levels of JAK2 and STAT3 were determined using reverse transcription-quantitative polymerase chain reaction analysis. β-actin was used as the internal control. The data are presented as the mean ± standard deviation of three independent experiments. ▲ P<0.05 and # P<0.01 vs. healthy group; ■ P<0.01, vs. entecavir group; °P<0.01, vs. 10% mXCHD group. mXCHD, modified Xiaochaihu decoction; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The membranes were then exposed to the primary antibodies against JAK2 (1:2,000; cat. no. 17670-1-AP; ProteinTech Group, Inc.) and STAT3 (1:1,000; cat. no. 60199-1-lg; ProteinTech Group, Inc) overnight at 4°C. β-actin (1:1,000; cat. no. 60008-1-lg; ProteinTech Group, Inc) was used as an internal control for protein loading.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Standard Deviation, Modification

Journal: Molecular Medicine Reports

Article Title: Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2017.7561

Figure Lengend Snippet: Effect of mXCHD serum on the protein expression levels of JAK2 and STAT3 in HepG2.2.15 cells. Following treatment with mXCHD serum, entecavir serum and healthy control serum for 48 h, the cells were collected. The protein levels of JAK2 and STAT3 were determined using western blot analysis. β-actin was used as the internal control. (A) Representative images of the results ofwestern blot analysis. (B) Data are presented as the mean ± standard deviation of three independent experiments. Δ P<0.05 vs. healthy group. mXCHD, modifiedXiaochaihu decoction; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The membranes were then exposed to the primary antibodies against JAK2 (1:2,000; cat. no. 17670-1-AP; ProteinTech Group, Inc.) and STAT3 (1:1,000; cat. no. 60199-1-lg; ProteinTech Group, Inc) overnight at 4°C. β-actin (1:1,000; cat. no. 60008-1-lg; ProteinTech Group, Inc) was used as an internal control for protein loading.

Techniques: Expressing, Control, Western Blot, Standard Deviation

Journal: Experimental hematology

Article Title: Phosphorylated c-MPL tyrosine 591 regulates thrombopoietin-induced signaling

doi: 10.1016/j.exphem.2014.02.007

Figure Lengend Snippet: (A) Top: Western blot analysis of phosphorylation levels of JAK2, STAT5, Akt and ERK1/2 in Ba/F3-MPL wild-type and YSF cells stimulated with 10ng/mL rhTPO for 0, 15, 60 or 120 minutes. Bottom: densitometry of phospho-ERK1/2 blot (*P < 0.05; ***P < 0.001). Error bars represent ±SEM. (n=3). (B) Top: Western blot analysis of phosphorylation levels of JAK2, STAT5, Akt and ERK1/2 in Ba/F3-MPL wild-type and Y591F cells stimulated for 5 minutes with 0, 0.1, 1 or 10ng/mL rhTPO. Bottom: densitometry of phospho-ERK1/2 blot. Error bars represent ±SEM. (n=3). (C) Top: Detection of active, GTP-bound Ras in Ba/F3-MPL wild-type and Y591F cells. Bottom: Densitometry of GTP-bound Ras blot (*P < 0.05). Error bars represent ±SEM. (n=3). Densitometry was performed using ImageJ

Article Snippet: Phosphospecific and total JAK2 (phospho-JAK2 clone C80C3 and total JAK2 clone D2E12), STAT5 (phospho-STAT5 clone D47E7), Akt (phospho-AKT clone D9E), ERK1/2 (phosphor-ERK1/2 clone D13.14.43 and total ERK clone 137F5), SYK (phospho-SYK Y323, phospho-SYK Y352 and phospho-SYK Y525/526 clone C87C1 and total SYK clone D1I5Q) and SHP-1 (phospho-SHP-1 clone D11G5 and SHP-1 clone C14H6) in addition to total BTK antibodies were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

doi: 10.1371/journal.pone.0027397

Figure Lengend Snippet: (A) After cultured for 9 h in medium without Epo, UT7 cells were left untreated or treated with 5 µM etoposide (VP16) or 0.5 µM doxorubicin (DXR) for 4 hr in the absence or presence of 50 mU/ml Epo, as indicated. Cells were lysed and subjected to immunoblot analysis with anti-Jak2 antibody, followed by reprobing with anti-EpoR and anti-β-actin, as indicated. (B, C) After cultured for 3 h in medium without Epo, 32D/EpoR cells were treated for 5 h in the absence or presence of 100 mU/ml Epo, as indicated, with increasing concentrations of etoposide (C) or doxorubicin (D), as indicated. Cell lysates were analyzed by immunoblotting with antibodies against indicated proteins. (D) 32D/EpoR or parental 32Dcl3 cells, cultured in medium containing 10% WEHI conditioning medium as the source of IL-3, were washed out of cytokine for 1 h. Cells were further cultured with or without 5 µM etoposide (VP16) for 6 h, as indicated, and analyzed. (E, F) After cultured for 9 h in medium without Epo, UT7/Jak2-V617F cells were treated for 1 h with or without 2 µM JakI-1. Cells were subsequently treated with increasing concentrations of etoposide (E; 0, 1, 2, 5 µM) or doxorubicin (D; 0, 0.1, 0.2, 0.5 µM), as indicated, and analyzed. (G) UT7/Jak2-V617F cells starved from Epo were pretreated with indicated concentrations of AG490 for 1 h. Cells were then treated with 5 µM etoposide or 0.5 µM doxorubicin for 6 h, as indicated, and analyzed.

Article Snippet: A rabbit antibody against Jak2 (06–255) and a mouse monoclonal antibody against phosphotyrosine (4G10) were purchased from Millipore (Billerica, MA, USA).

Techniques: Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

doi: 10.1371/journal.pone.0027397

Figure Lengend Snippet: (A) After cultured for 9 h in medium without Epo, UT7 cells were pretreated for 1 h with 50 µM LY294002 (PI3K-I) or 50 µM PD98059 (MEK-I), as indicated, or left untreated. Cells were subsequently treated with or without 10 µM etoposide (VP16) for 4 h, as indicated, in the presence of 20 mU/ml Epo or in its absence (Epo -). Cells were lysed and subjected to immunoblot analysis with anti-Jak2 antibody, followed by sequential reprobing with anti-phospho-GSK3α/β-S9/21 (GSK3β-P), anti-GSK3β, anti-β-actin, as indicated. (B) 32D/Akt-myr (Akt-myr) as well as control 32D/RevTRE (Cont.) cells were cultured for 24 h with 1 µg/ml doxycycline to induced the expression of Akt-myr in 32D/Akt-myr cells and subsequently washed out of WEHI conditioning medium for 12 h. Cells were then pretreated for 1 h with 1 µM JakI-1 or 10 µM LY294002 (PI3K-I), as indicated, or left untreated. Cells were finally treated with or without 10 µM etoposide (VP16), as indicated, for 4 h before analysis with indicated antibodies. (C) After cultured for 9 h in medium without Epo, UT7 cells were pretreated for 1 h with 10 µM SB216763 (SB216), 40 mM LiCl, or okadaic acid at 100 nM (OA100) or 200 nM (OA200), as indicated, or left untreated. Cells were subsequently treated with or without 10 µM etoposide (VP16) for 4 h, as indicated, and analyzed. (D) 32DE/STAT5A1*6 (STAT5A1*6) or control 32DE/pMX (Cont.) cells were pretreated for 1 h with 1 µM JakI-1 or 50 µM LY294002 (PI3K-I), as indicated, or left untreated in the absence of Epo. Cells were further treated with or without 5 µM etoposide (VP16) for 6 h, as indicated, before analysis. (E) 32DE/STAT5A1*6 (STAT5A1*6) or control 32DE/pMX (Cont.) cells were cultured overnight in the absence of Epo. Cells were lysed and subjected to immunoprecipitation of p85. Immunoprecipitates were analyzed by immunoblotting. (F) After cultured for 12 h in medium without Epo, UT7/Jak2-V617F cells were pretreated for 1 h with 50 µM LY294002 (PI3K-I), 2 µM JakI-1, 40 mM LiCl, or 10 µM MG132, as indicated, or left untreated as control (Cont.). Cells were subsequently treated with or without 5 µM etoposide (VP16), as indicated, for 6 h and analyzed.

Article Snippet: A rabbit antibody against Jak2 (06–255) and a mouse monoclonal antibody against phosphotyrosine (4G10) were purchased from Millipore (Billerica, MA, USA).

Techniques: Cell Culture, Western Blot, Control, Expressing, Immunoprecipitation

Journal: PLoS ONE

Article Title: DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

doi: 10.1371/journal.pone.0027397

Figure Lengend Snippet: (A) 32D/EpoR cells deprived of Epo for 2 h were pretreated with 10 µM MG132 or left untreated as control, as indicated for 1 h in the absence of Epo. Cells were then treated for 6 h with or without 5 µM etoposide, as indicated. Cells were lysed and subjected to immunoblot analysis using indicated antibodies. (B) 293T cells were transfected on 6-well plate with 0.1 µg of pRK5-Ubiquitin-WT and 0.002 µg of pRK5-Jak2-Wt (Wt) or pRK5-Jak2-KE (KE) along with 0.1 µg of pXM-EpoR-Wt or empty plasmid, as indicated. Two days after transfection, cells were lysed, and Jak2 was immunoprecipitated. Immunoprecipitates were analyzed by immunoblotting using antibodies against polyubiquitin (poly-Ubi), Jak2, and Jak2 phosphorylated on Y1007 (Jak2-PY), as indicated. The vertical line indicates the smeary pattern characteristic of ubiquitination. (C) 293T cells were transfected on 6-well plate with 0.3 µg of pRevTRE-Jak2V617F and 0.2 µg of pTet-On along with 0.2 µg of pRK5-Ubiquitin-WT (Ubi), 0.5 µg of pRevTRE-c-Cbl (c-Cbl), or 0.5 µg of pRevTRE-Cbl-b (Cbl-b), as indicated. The amounts of plasmid DNA transfected were equalized by adding pRevTRE. Two days after transfection, cells were lysed, and lysates were analyzed by immunoblotting. The position of unmodified Jak2-V617F and those of ubiquitinated Jak2-V617F are indicated by asterisks and arrows, respectively. (D) Ton.32D/Flt3-Wt (Cont.) and Ton.32D/Flt3-Wt/c-Cbl-RQ/Cbl-b-CA (c-Cbl-RQ/Cbl-b-CA) cells, cultured in doxycycline-containing medium, were cultured for 3 h with or without 5 µM LY294002 (LY), as indicated, in the absence of IL-3. Cells were further treated for 6 h with 10 µM etoposide (VP16) or 1 µM doxorubicin (DXR), as indicated, and lysed. Cell lysates were analyzed by immunoblotting.

Article Snippet: A rabbit antibody against Jak2 (06–255) and a mouse monoclonal antibody against phosphotyrosine (4G10) were purchased from Millipore (Billerica, MA, USA).

Techniques: Control, Western Blot, Transfection, Ubiquitin Proteomics, Plasmid Preparation, Immunoprecipitation, Cell Culture

Journal: PLoS ONE

Article Title: DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

doi: 10.1371/journal.pone.0027397

Figure Lengend Snippet: (A) UT7/Jak2-V617F cells were cultured for 12 h with 0.5 µM etoposide (VP16), 0.5 µM JakI-1, 25 µM LY294002, and 1 µM GSK3I-5, as indicated in the absence of Epo, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (B) UT7/Jak2-V617F cells were cultured for 6 h with 5 µM etoposide (VP16), 1 µM JakI-1, and 100 µM Boc-d-fmk, as indicated, in the absence of Epo and analyzed for the cellular DNA content. (C, D) 32D/EpoR cells deprived of Epo for 2 h were pretreated for 1 h with 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated for 5 h with or without 5 µM etoposide (VP16) or 0.5 µM doxorubicin (DXR), as indicated. Cells were lysed and analyzed by immunoblotting. (E) UT7/Jak2-V617F cells, cultured without Epo for 12 h, were treated with 1 µM JakI-1 and 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated with or without 5 µM etoposide (VP16), as indicated, for 6 h and analyzed.

Article Snippet: A rabbit antibody against Jak2 (06–255) and a mouse monoclonal antibody against phosphotyrosine (4G10) were purchased from Millipore (Billerica, MA, USA).

Techniques: Cell Culture, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

doi: 10.1371/journal.pone.0027397

Figure Lengend Snippet: (A) UT7/Jak2-V617F cells, cultured without Epo, were treated for 7 h with or without 1 µM JakI-1, as indicated, with 1 µM etoposide (VP16) added for indicated times before harvest. Cells were then analyzed for activation of Bax or caspase-3 by flow cytometry. Percentages of cells with activated Bax or caspase-3 are plotted. FSC: forward scatter. (B) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with 1 µM JakI-1, 100 µM Boc-d-fmk, or 1 µM GSK3I-5, as indicated. Cells were further treated for 6 h with or without 5 µM VP16 and analyzed for loss of mitochondrial membrane potential and activation of Bax or caspase-3, as indicated, by flow cytometry. (C) 32D/EpoR cells were deprived of Epo and pretreated with 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated for 5 h with or without etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Jak2 (06–255) were analyzed by immunoblotting using anti-Jak2 (M-126) and anti-Jak2 (C-20), as indicated. The numbers of cells treated with etoposide were 2.5 times that of cells untreated. An arrow indicates the 100-kDa band. (D) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with or without 1 µM JakI-1, as indicated. Cells were then treated for 8 h with or without 5 µM etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Jak2 (06–255) were analyzed by immunoblotting using indicated antibodies. The number of cells treated with etoposide was 2 times that of cells untreated. (E) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with 1 µM JakI-1 and 20 µM Boc-d-fmk (B-d-f), as indicated. Cells were then treated for 6 h with or without 5 µM etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Flag was were analyzed by immunoblotting using anti-Jak2 (06–255). The numbers of cells treated with etoposide were 2.5 times that of cells untreated.

Article Snippet: A rabbit antibody against Jak2 (06–255) and a mouse monoclonal antibody against phosphotyrosine (4G10) were purchased from Millipore (Billerica, MA, USA).

Techniques: Cell Culture, Activation Assay, Flow Cytometry, Membrane, Western Blot