Journal: Inflammation Research

Article Title: Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo

doi: 10.1007/s00011-014-0782-9

Figure Lengend Snippet: IC 50 and Ki values of JAK inhibitors in enzyme assays

Article Snippet: Recombinant kinase domains of human JAK1 (850–end) and Tyk2 (871–end) were purchased from Carna Biosciences Inc. (Kobe, Japan), and those of JAK2 (808–end) and JAK3 (781–end) were from Millipore Corporation (Billerica, MA).

Techniques:

Journal: Inflammation Research

Article Title: Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo

doi: 10.1007/s00011-014-0782-9

Figure Lengend Snippet: IC 50 values in cytokine signaling

Article Snippet: Recombinant kinase domains of human JAK1 (850–end) and Tyk2 (871–end) were purchased from Carna Biosciences Inc. (Kobe, Japan), and those of JAK2 (808–end) and JAK3 (781–end) were from Millipore Corporation (Billerica, MA).

Techniques:

Journal: JOR spine

Article Title: M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway.

doi: 10.1002/jsp2.1290

Figure Lengend Snippet: FIGURE 2 Oncostatin M (OSM) can promote the ossification of ligament flavum (LF) cells by activating the JAK2/STAT3 signaling pathway. (A) Upstream regulator analysis was used to evaluate the expression degree of each signaling pathway in osteogenic differentiation of LF cells. (B) Upregulated signal molecules downstream of OSM were analyzed by upstream regulator analysis. (C–F) Upstream regulator analysis was used to analyze the significance of the top 10 factors in each category of cytokines, kinases, transcription factors, and signaling pathway inhibitors. (G) Upstream regulator analysis was used to investigate the significance of growth factors other than OSM during osteogenic differentiation of LF cells.

Article Snippet: To investigate the effect of the JAK2/STAT3 signaling pathway on osteogenic differentiation of LF cells, the JAK2 inhibitor AZD1480 (10 μM, Santa Cruz Biotechnology), STAT3 inhibitor Stattic (5 μM, Santa Cruz Biotechnology), or blank control (DMSO) was added to the osteogenic differentiation medium with 25 ng/mL recombinant OSM protein to induce LF cells.

Techniques: Expressing

Journal: JOR spine

Article Title: M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway.

doi: 10.1002/jsp2.1290

Figure Lengend Snippet: FIGURE 4 Oncostatin M (OSM) promotes osteogenic differentiation of ligamentum flavum (LF) cells through the JAK2/STAT3 signaling pathway. (A–C) Expression of OSMR, GP130, P-JAK2, P-STAT, and osteogenic genes was detected by western blot (WB) and qPCR after 7 days of OSM cytokine induction (n = 3), two-tailed Student's t-test. (D–G) Expression of OSMR and GP130 was evaluated after knockdown in LF cells (n = 3), two-tailed Student's t-test. (H, I) LF cells with knockdown of OSMR and GP130, and stimulation with OSM. WB and qPCR were performed to evaluate expression of P-JAK2, P-STAT3, and osteogenic differentiation genes (n = 3), one-way ANOVA analysis. (J, K) In the presence of AZD1480 and Stattic, expression of P-JAK2, P-STAT3, and osteogenic genes was detected by WB and qPCR after 7 days of OSM induction in LF cells (n = 3), one-way ANOVA analysis. All data were presented as means ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To investigate the effect of the JAK2/STAT3 signaling pathway on osteogenic differentiation of LF cells, the JAK2 inhibitor AZD1480 (10 μM, Santa Cruz Biotechnology), STAT3 inhibitor Stattic (5 μM, Santa Cruz Biotechnology), or blank control (DMSO) was added to the osteogenic differentiation medium with 25 ng/mL recombinant OSM protein to induce LF cells.

Techniques: Expressing, Western Blot, Two Tailed Test, Knockdown, Standard Deviation

Journal: JOR spine

Article Title: M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway.

doi: 10.1002/jsp2.1290

Figure Lengend Snippet: FIGURE 5 M1 macorphages (Mφs) can secrete oncostatin M (OSM) and induce osteogenic differentiation of ligamentum flavum (LF) cells via the JAK2/STAT3 signaling pathway. The cytokine OSM binds to the receptor OSMR/GP130, stimulating activation and phosphorylation of JAK2. Phosphorylated JAK2 subsequently stimulates phosphorylation of downstream STAT3, causing STAT3 to form a dimer that migrates to the nucleus and binds to DNA to regulate gene transcription. Cyclooxygenase-2 inhibitors reduce OSM secretion by M1 Mφs. OSM neutralizing antibody can inhibit the binding of OSM to its receptor. Knockdown of OSMR/GP130 can obstruct signal transmission. AZD1480 inhibits both expression and phosphorylation of JAK2. Stattic can block STAT3 activation and phosphorylation.

Article Snippet: To investigate the effect of the JAK2/STAT3 signaling pathway on osteogenic differentiation of LF cells, the JAK2 inhibitor AZD1480 (10 μM, Santa Cruz Biotechnology), STAT3 inhibitor Stattic (5 μM, Santa Cruz Biotechnology), or blank control (DMSO) was added to the osteogenic differentiation medium with 25 ng/mL recombinant OSM protein to induce LF cells.

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Knockdown, Transmission Assay, Expressing, Blocking Assay

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: Multiscale analysis identifies α‐Hederin as a JAK/STAT3‐targeting compound for OC. (A) 3D Principal component analysis (PCA). PCA plot was generated using the 2500 genes with the highest variance across samples. Normal tissues are represented by orange points, while OC samples are indicated by blue points. (B) Volcano plot showing differentially expressed genes between OC and normal tissues. (C) Boxplot showing increased mRNA expression of IL‐6 in OC tissues compared to normal tissues, based on TCGA and GTEx datasets. (D) GSEA indicating significant enrichment of the IL‐6/JAK/STAT3 signaling pathway in OC. (E) Uniform Manifold Approximation and Projection (UMAP) plot of 51 786 single cells from 11 epithelial ovarian cancer (EOC) patients ( GSE165897 ), color‐coded by patient identity. (F) Expression levels of IL6, IL6ST, JAK1, and STAT3 across single‐cell populations. (G) UMAP plots show the distribution of cells before and after NACT treatment. (H) JAK1 expression levels before and after NACT treatment. (I) Workflow of structure‐based virtual screening of 2908 natural compounds targeting JAK1 and JAK2, followed by cytotoxicity validation in OC and normal ovarian epithelial cells. (J) Dose‐response curves quantifying viability of OC cells upon drug treatment for 48 h. The code names of drugs are listed on the right. (K) Dose‐response curves quantifying viability of ovarian epithelial cells upon drug treatment for 48 h. (L) Binding affinity measurements of α‐Hederin and JAK1 as measured via MST thermophoresis curve analysis. (M) Binding affinity measurements of α‐Hederin and JAK2 as measured via MST thermophoresis curve analysis. (N) Schematic structures of JAK1. Molecular docking results of α‐Hederin (green) with JAK1 (blue). The docking sites of α‐Hederin on JAK1 were highlighted in magenta. (O) Schematic structures of JAK2. Molecular docking results of α‐Hederin (green) with JAK2 (blue). The docking sites of α‐Hederin on JAK2 were highlighted in magenta. (P) Venn diagram displaying α‐Hederin targets (pink) and OC‐associated genes (yellow). The overlapping regions indicate common targets. (Q) KEGG analysis highlighted the top 20 pathways with significant enrichment. Then red box indicated the JAK/STAT3 signaling pathway. (R)The schematic diagram of the drug‐target gene network was visualized using Cytoscape software.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Generated, Expressing, Biomarker Discovery, Binding Assay, Software

Journal: Advanced Science

Article Title: Discovery of Natural Compound α‐Hederin via Large‐Scale Screening as a Targeted JAK/STAT3 Inhibitor for Ovarian Cancer Therapy

doi: 10.1002/advs.202417278

Figure Lengend Snippet: α‐Hederin directly binds to JAK1/2 and inhibits STAT3 phosphorylation and nuclear translocation. (A,B) IF staining of p‐STAT3 and statistical analysis of fluorescence intensity. Scale bar: 10 µ m . (C) Heatmap showing relative mRNA expression of STAT3 downstream targets (MYC, CCND1, BIRC5, BCL2, VEGFA, TWIST1, MMP2, and MMP9) following α‐Hederin treatment, measured by qRT‐PCR and normalized to GAPDH. (D) Western blot analysis of total and phosphorylated STAT3, JAK1, JAK2, JAK3, and SRC in SKOV‐3 cells treated with α‐Hederin (5 or 10 µ m ) or DMSO. Statistical analysis is presented. (E,F) Kinase assay to examine the effect of α‐Hederin on JAK1 (E) and JAK2 (F) kinase activity. (G,H) The competitive binding relationship between α‐Hederin and ATP was confirmed using a pull‐down assay. (I,J) In vitro kinase assays were performed using bacterial‐purified His‐STAT3 and the active JAK1 (I) and JAK2 (J) kinase. The amount of α‐Hederin in the reaction is indicated. (K) DARTS (drug affinity responsive target stability) assay showing α‐Hederin‐mediated stabilization of JAK1 and JAK2 proteins in SKOV‐3 lysates. (L) Western blot analysis of p‐STAT3 and total STAT3 in SKOV‐3 cells with sgCtrl, sgJAK1, sgJAK2, or sgJAK1+sgJAK2, treated or not with 5 µ m α‐Hederin. Bottom panel: quantification of p‐STAT3/STAT3 ratio. Data are presented as mean ± SD from at least three independent experiments. Statistical significance was determined by unpaired two‐tailed Student's t ‐test for two‐group comparisons and one‐way ANOVA for comparisons among multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Active human JAK1 and JAK2 kinase, obtained from Signal Chem (Canada), was diluted to a final concentration of 0.1 μg mL −1 in Kinase Dilution Buffer III (Signal Chem, K23‐09).

Techniques: Phospho-proteomics, Translocation Assay, Staining, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot, Kinase Assay, Activity Assay, Binding Assay, Pull Down Assay, In Vitro, Purification, Stability Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Single‐Cell Transcriptomics Reveals ITGA2‐Mediated Metabolic Reprogramming and Immune Crosstalk in Pediatric Thyroid Carcinogenesis

doi: 10.1002/advs.202504088

Figure Lengend Snippet: ITGA2 hi ‐PTC induces macrophage polarization. a) Circle plots show cell‐cell interaction strength differences of ITGA2 hi ‐PTC and SPP1 hi ‐Macro. b) Schematic diagram of in vitro chemotaxis of THP‐1 by ITGA2 via TPC‐1/THP‐1 conditioned media culture model. c,d,e) THP‐1 cells were differentiated by PMA pre‐treatment for 24 h, then cocultured with TPC‐1 cells for 2 days. Transwell assays were used to detect and observe the chemotaxis of THP‐1 after TPC‐1 cells were overexpressed‐ITGA2, knockdown‐ITGA2, or co‐treated with rITGA2. f) Schematic depiction of experimental design for macrophage phenotype polarization by ITGA2 hi ‐PTC, ① THP‐1 cells were stimulated with PMA to differentiate into M0,② PTC cells are allowed to generate ITGA2, ③ M0 macrophages are exposed to ITGA2. g,h,i) The expression levels of surface markers in M0‐differentiated THP‐1 cells exposed to TPC‐1 cells with overexpressed ITGA2 using flow cytometry. j) ELISA detected the level change of IL‐1α, IL‐1β, TNF‐α, IL‐4, IL‐10, and TGF‐β in THP‐1 cell supernatant after exposed to TPC‐1 cells with overexpressed ITGA2. k,l,m) The expression levels of surface markers in M0‐differentiated THP‐1 cells exposed to rITGA2 using flow cytometry. n) ELISA detected the level change of IL‐1α, IL‐1β, TNF‐α, IL‐4, IL‐10, and TGF‐β in THP‐1 cell supernatant after exposed to rITGA2. o) Heatmaps show the activity intensity of macrophage subtypes in selected hallmark pathways by GSVA analysis, Z‐score normalized: range −1–1. p) Representative immunoblot reveals the expression change of ITGA2, JAK2, STAT3, p‐JAK2, and p‐STAT3 in THP‐1 cells exposed to rITGA2. q)Schematic model depicting the mechanism by which ITGA2 hi ‐PTC cells drive M2 macrophage polarization via JAK2‐STAT3 signaling activation.​The results are shown as the mean ± SEM of three independent experiments. Two‐sided student's t test was used to estimate statistical significance, * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance. ITGA2 hi ‐PTC, ITGA2‐high expressing papillary thyroid carcinoma cells. THP‐1, Human leukemia monocytic cell line. PMA, Phorbol 12‐myristate 13‐acetate. rITGA2, Recombinant ITGA2 protein. M0, Non‐ polarized macrophage. ELISA, Enzyme‐linked immunosorbent assay. GSVA, Gene set variation analysis. p‐JAK2, Phosphorylated JAK2. p‐STAT3, Phosphorylated STAT3.

Article Snippet: Post‐transfer, the membranes were blocked with a blocking solution consisting of 5% bovine serum albumin (BSA) and 5% milk in Tris‐buffered saline with Tween 20 (TBST) at ambient temperature for a duration of 2 h. The specific primary antibodies employed included: β‐actin (Abcam, ab8227, dilution 1:5000), ITGA2 (Abcam, ab133557, dilution 1:10000), GLUT1 (Proteintech, 829‐1‐AP, dilution 1:4000), JAK2 (Proteintech, 17670‐1‐AP, dilution 1:600), pJAK2 (Cell Signaling Technology, 4406S, dilution 1:1000), STAT3 (Cell Signaling Technology, 9139S, dilution 1:1000), pSTAT3 (Cell Signaling Technology, 9145S, dilution 1:2000), and GAPDH (Proteintech, HRP‐60004, dilution 1:10000).

Techniques: In Vitro, Chemotaxis Assay, Knockdown, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Activation Assay, Recombinant

Journal: JAK-STAT

Article Title: A rapid conformational rearrangement of STAT1 dimers is required for termination rather than for amplification of interferon-γ signaling

doi: 10.4161/jkst.23576

Figure Lengend Snippet: Figure 5. Phosphorylated STAT1-F364A is partially resistant against inactivation by Tc45 phosphatase. ( A–D ) In vitro phosphorylation assays demonstrate unaltered tyrosine phosphorylation of STAT1-F364A. Whole cell extracts from reconstituted U3A cells expressing either STAT1-WT or -F364A (10 μl in each reaction) were incubated with 40 ng of recombinant JAK2 kinase ( A and B ) or 20 ng of EGF receptor [EGFR, ( C and D )] and incorporation of phosphate in STAT1 was monitored with time by means of western blotting. Statistical analyses revealed no significant difference in the phosphorylation kinetics between wild-type and mutant STAT1 (p > 0.05). ( E and F ) STAT1-F364A is partially protected against the attack of the inactivating phosphatase, as revealed by an in vitro dephosphorylation assay. Extracts from IFNγ-prestimulated U3A cells (10 μl each) were incubated with 2 U of the STAT1-specific Tc45 phosphatase and tyrosine dephosphorylation was followed for 30 min. Shown are a representative western blot result ( E ) and a quantitative depiction ( F ) of the specific tyrosine phosphorylation (phosphotyrosine signal divided by total STAT1 signal) with bars expressing means and standard deviations. Significant differences between wild-type and mutant STAT1 from five independent experiments are indicated with asterisks (p = 0.034 and p = 0.017, respectively). ( G ) An electrophoretic mobility shift assay demonstrates the exchange of monomers between STAT1 dimers. Shown is a representative gelshift result using cellular extracts from U3A cells expressing either GFP-tagged or untagged STAT1 bound to M67 DNA. The identity of the bands corresponding to STAT1 (marked with arrowheads) was confirmed by the absence of or reduction in DNA-binding activity in IFNγ-stimulated Y701F- (lane 1) and unstimulated WT-expressing cells (lane 2) as well as from supershift reactions using either a STAT3- (lanes 3 and 5) or STAT1-recognizing antibody (lanes 4 and 6). In lanes 7–10, similar amounts of GFP-tagged and untagged homodimers were either immediately mixed and incubated together for 45 min (lanes 8 and 10) or incubated separately for 45 min before being loaded together onto the gel (lanes 7 and 9). The asterisk at the right margin marks a non-specific band.

Article Snippet: For in vitro phosphorylation assays, 10 μl of whole cell extract from STAT1-reconstituted U3A cells were mixed with 10 μl of kinase buffer containing 50 mM Hepes, pH 7.4, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 µM vanadate, 10 mM DTT, 0.1 mM ATP and either 40 ng of recombinant Janus kinase 2 (JAK2, obtained from Enzo Life Science) or 20 ng of epidermal growth factor receptor (EGFR, obtained from US Biological).

Techniques: In Vitro, Expressing, Incubation, Recombinant, Western Blot, Mutagenesis, De-Phosphorylation Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

Journal: Endocrine-Related Cancer

Article Title: Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway

doi: 10.1677/erc-06-0027

Figure Lengend Snippet: Figure 3 Leptin activates phosphorylations of JAK2, Akt, and ERK1/2 in hepatic cells. After 1-day serum deprivation, leptin was added into the serum-free medium of Hep3B for 30 min (A) or Chang liver for 15 min (B) with the increasing concentrations as indicated, and then the protein amounts of phosphorylated forms of JAK2 (p-JAK2), Akt (p-Akt), or ERK1/2 (p-ERK1/2) were detected with western blotting analyses. The same blots were stripped and reprobed with antibodies specific for total proteins of JAK2, Akt, or ERK1/2. Similar western blotting analyses were carried out with the cell lysates of (C) Hep3B and (D) Chang liver treated with 250 ng/ml leptin for the indicated time-courses. Data represent three independent experiments. The reprobed b-actin was used as an alternative internal control. Leptin acutely and dose-dependently induces activations of JAK2, Akt, and ERK1/2 in human malignant and non-malignant hepatocytes.

Article Snippet: Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-b-actin antibody (Sigma Chemical Co.), TGF-b (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and www.endocrinology-journals.org monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.

Techniques: Western Blot, Control

Journal: Endocrine-Related Cancer

Article Title: Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway

doi: 10.1677/erc-06-0027

Figure Lengend Snippet: Figure 5 Leptin triggers JAK2-linked PI3K/Akt and MEK/ERK1/2 signaling pathways. After 1-day serum deprivation and then 30-min pre-treatment of vehicle (K), 40 mM AG490 (AG), 250 nM wortmannin (Wort), or 10 mM U0126 (U0), (A) Hep3B and (B) Chang liver were treated with vehicle (K) or 250 ng/ml leptin (C) in serum-free media for 30 and 15 min respectively. Following, the protein amounts of phosphorylated forms of JAK2 (p-JAK2), Akt (p-Akt), or ERK1/2 (p-ERK1/2) were detected with western blotting analyses. The same blots were stripped and reprobed with antibodies specific for total proteins of JAK2, Akt, or ERK1/2. The reprobed b-actin was used as an alternative internal control. Alternative inhibitors 25 mM LY294002 (LY) and 20 mM PD98059 (PD) for PI3K/Akt and MEK/ERK1/2 respectively, were tested in the similar experiments in Hep3B (C). Data represent three independent experiments. Leptin activates a JAK2-initiated signaling cascade comprising PI3K/Akt and MEK/ERK1/2 in order of occurrence.

Article Snippet: Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-b-actin antibody (Sigma Chemical Co.), TGF-b (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and www.endocrinology-journals.org monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.

Techniques: Protein-Protein interactions, Western Blot, Control

Journal: Endocrine-Related Cancer

Article Title: Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway

doi: 10.1677/erc-06-0027

Figure Lengend Snippet: Figure 9 Leptin passes through a JAK2–PI3K/Akt– MEK/ERK1/2 signaling cascade to partially reverse the TGF- b1-reduced Bcl-2/Bax ratio and thus prevent HCC apoptosis. After 24 h, Hep3B cells were seeded in 100 mm Petri dish and maintained in serum-free media, cells were treated without (K) or with (C) indicated concentrations of leptin, together without (K) or with (C) 5 ng/ml TGF-b1 in combination without (K) or with (C) 40 mM AG490 (A), 250 nM wortmannin or 10 mM U0126 (B) in serum-free media for 48 h. Subsequently, their cell lysates were subjected to western blotting analyses for detecting the cleavage of PARP and the protein amounts of Bcl-2 and Bax using b-actin as an internal control. The active PARP (116 kDa) together with the larger fragment of cleaved PAPR (89 kDa) and the calculated Bcl/Bax ratio from each treatment are indicated. Activations of JAK2, PI3K/Akt, and MEK/ERK1/2 are essential for mediating leptin inhibition on TGF-b1-induced Bax expression and resulting Hep3B apoptosis.

Article Snippet: Human recombinant leptin, epidermal growth factor (EGF), tyrphostin AG490, U0126, wortmannin, LY294002, PD98059, monoclonal anti-b-actin antibody (Sigma Chemical Co.), TGF-b (R&D Systems Inc., Minneapolis, MN, USA), polyclonal antibodies against phospho-JAK2 (Upstate, Charlottesville, VA, USA), Bax, PARP, phospho-Akt, or phospho-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), JAK2 or ERK1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and www.endocrinology-journals.org monoclonal antibody against Akt, Bcl-2, or cyclin D1 (BD Pharmingen, Palo Alto, CA, USA) were commercially obtained.

Techniques: Western Blot, Control, Inhibition, Expressing

Journal: Cancer cell

Article Title: Therapeutic Ablation of Gain-of-Function Mutant p53 in Colorectal Cancer Inhibits Stat3-Mediated Tumor Growth and Invasion

doi: 10.1016/j.ccell.2018.07.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For immunoblotting equal amounts of protein lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto nitrocellulose membranes (Millipore), blocked with 5% milk dissolved from powder and probed with the following antibodies: p53 (CM5, Vector Laboratories) for murine p53, p53 (DO-1, Santa Cruz sc-126) for human p53, Hsc70 (B-6, Santa Cruz sc-7298), phospho-Y705 Stat3 XP (Abcam, ab76315), total Stat3 (Santa Cruz, sc-482) or total Stat3 (79D7) (Cell Signaling, #4904), phospho-Jak2 (Tyr1007/1008) (C80C3) (Cell Signaling #3776), Jak2 (Abcam, ab108596), cyclinD1 (Abcam, ab134175) and Actin (Abcam, ab8227).

Techniques: Recombinant, Mutagenesis, Cell Culture, Negative Control, Plasmid Preparation, Clone Assay, Software

Journal: Clinical Cancer Research

Article Title: SD-1029 Inhibits Signal Transducer and Activator of Transcription 3 Nuclear Translocation

doi: 10.1158/1078-0432.ccr-06-1330

Figure Lengend Snippet: Fig. 5. SD-1029 blocksJak2 phosphorylation. A, SD-1029 inhibits phosphorylation ofJak2 in cell lines. OVCAR8TR cells and MDA-MB-468 cells were treated with 10 Amol/L of SD-1029 in a time-dependent manner or with SD-1029 for 24 hours in a dose-response manner. ForWestern blot analysis, 25 Ag of cell-free extracts was subjected to immunoblotting with specific antibodies toJak2 and Jak2 as described in Materials and Methods. B, SD-1029 directly inhibitsJak2 kinase autophosphorylation in vitro. Equal amounts ofJak2 recombinant protein were divided and preincubated in the presence or absence of various concentrations of SD-1029, AG490, or DMSO control for1hour. Following the addition of1mmol/L of ATP and an additional 60 minutes of incubation at room temperature, the reaction was halted using a stop buffer. Jak2 autophosphorylation was visualized as described in Materials and Methods.

Article Snippet: The Jak2 autophosphorylation kinase assay was done using recombinant Jak2 protein (Upstate Biotechnology).

Techniques: Phospho-proteomics, Western Blot, In Vitro, Recombinant, Control, Incubation