total jak2 antibody  (Cell Signaling Technology Inc)

Journal: Neurotherapeutics

Article Title: Ruxolitinib-dependent reduction of seizure load and duration is accompanied by spatial memory improvement in the rat pilocarpine model of temporal lobe epilepsy

doi: 10.1016/j.neurot.2024.e00506

Figure Lengend Snippet: Effect of RUXO on SE-mediated JAK2 activation. SE-mediated increased in phosphorylation of JAK2 was assessed in hippocampus of Sham (n ​= ​3), DMSO-treated (n ​= ​3) and RUXO-treated (n ​= ​3) rats at 30 ( a ) and 60 ( b ) min after SE. Phosphorylation levels were obtained upon normalization with total JAK2. Data are expressed as % of Sham and shown as box and whiskers (min-max range). Due to the short time interval between sample collections, and the absence of intra-group significant differences recorded for pJAK2 levels at 30 and 60 ​min (see Supplementary Fig. 3 ), data were combined for each experimental group and statistical analysis performed by Kruskal-Wallis test followed by Dunn's post-hoc test.

Article Snippet: Subsequently, membranes were incubated overnight at 4 °C in 5 % bovine serum albumin/TBS-T with primary antibodies raised against the following targets: Cyclin D1 [Cell Signaling Technologies (CST) # #2978; 1:500]; STAT3 phosphorylated at Tyr705 (anti-pSTAT3; CST #9131S ; 1:1000) and total STAT3 (CST #4904S ; 1:6000); JAK2 phosphorylated at Tyr1007/1008 (anti-pJAK2; CST; 1:400) and total JAK2 (1:2000); ICER (Millipore/Sigma #SAB2500271; 1:100).

Techniques: Activation Assay, Phospho-proteomics

Journal: Chinese Journal of Analytical Chemistry

Article Title: Active substances and molecular mechanisms of Astragali Radix and Angelicae Sinensis Radix against idiopathic pulmonary fibrosis effects by network pharmacology and in vitro experiments

doi: 10.1016/j.cjac.2024.100397

Figure Lengend Snippet: Fig. 7. Molecular docking results of main components of AR and ASR. (A) Quercetin - IL-6; (B) magnolol - JAK2; (C) riligustilide - JAK2; (D) coniferyl ferulate - STAT3; (E) valerophenone - STAT3.

Article Snippet: The primary antibodies in western blot were as follows: Anti ‐α- MA (catalog number: 19,245), anti ‐STAT3 (catalog number: 12640S), nti ‐phospho ‐STAT3 (catalog number: 9145S), anti ‐total ‐JAK2 (catalog umber: 3230S), anti ‐phospho ‐JAK2 (catalog number: 3771S) antibodes were obtained from Cell Signaling Technology (Danvers, MA).

Techniques:

Journal: Chinese Journal of Analytical Chemistry

Article Title: Active substances and molecular mechanisms of Astragali Radix and Angelicae Sinensis Radix against idiopathic pulmonary fibrosis effects by network pharmacology and in vitro experiments

doi: 10.1016/j.cjac.2024.100397

Figure Lengend Snippet: Fig. 9. Effects of quercetin and magnolol on the JAK2/STAT3 signaling pathway in the A549 cells. Data are expressed as mean ± tan- dard error of the mean (SEM) ( n = 3/group). # p < 0.05 vs. Con group. ## p < 0.01 vs. Con group. ∗ p < 0.05 vs. IL-6 group. ∗ ∗ p < 0.01 vs. IL-6 group. △p < 0.05 vs. Magnolol group. △△p < 0.01 vs. Magnolol group.

Article Snippet: The primary antibodies in western blot were as follows: Anti ‐α- MA (catalog number: 19,245), anti ‐STAT3 (catalog number: 12640S), nti ‐phospho ‐STAT3 (catalog number: 9145S), anti ‐total ‐JAK2 (catalog umber: 3230S), anti ‐phospho ‐JAK2 (catalog number: 3771S) antibodes were obtained from Cell Signaling Technology (Danvers, MA).

Techniques:

Journal: Chinese Journal of Analytical Chemistry

Article Title: Active substances and molecular mechanisms of Astragali Radix and Angelicae Sinensis Radix against idiopathic pulmonary fibrosis effects by network pharmacology and in vitro experiments

doi: 10.1016/j.cjac.2024.100397

Figure Lengend Snippet: Fig. 10. The mRNA expression of the JAK2/STAT3 signaling path- way in the A549 cells. (A) HIF-1 𝛼mRNA expression. (B) 𝛼-SAM mRNA expression. Data are expressed as mean ± SEM ( n = 6/group) #p < 0.05 vs. Con group. ## p < 0.01 vs. Con group. ∗ p < 0.05 vs. IL-6 group. ∗ ∗ p < 0.01 vs. IL-6 group. △p < 0.05 vs. Magnolol group. △△p < 0.01 vs. Magnolol group.

Article Snippet: The primary antibodies in western blot were as follows: Anti ‐α- MA (catalog number: 19,245), anti ‐STAT3 (catalog number: 12640S), nti ‐phospho ‐STAT3 (catalog number: 9145S), anti ‐total ‐JAK2 (catalog umber: 3230S), anti ‐phospho ‐JAK2 (catalog number: 3771S) antibodes were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing

Journal: Cancers

Article Title: KLC1-ROS1 Fusion Exerts Oncogenic Properties of Glioma Cells via Specific Activation of JAK-STAT Pathway

doi: 10.3390/cancers16010009

Figure Lengend Snippet: Expression of KLC1-ROS1 fusion specifically activates the JAK-STAT pathway in glioma cells. ( a ) Schematic illustration of the domain structure of wild-type ROS1 (ROS1), wild-type KLC1 (KLC1), and KLC1-ROS1 fusion. The breakpoints of ROS1 (exon35) and KC1 (exon9) are indicated by an arrowhead with the corresponding amino acid (aa) positions. ( b , c ) Plasmids encoding the empty vector, FLAG -tagged wild-type (WT) ROS1 , and FLAG -tagged KLC1-ROS1 fusion were stably expressed in A172 and U343MG human glioblastoma cell lines (( b ) A172 cells, ( c ) U343MG cells). The cell lysates were analyzed by immunoblotting using the indicated antibodies. Primary anti-beta actin antibody and anti-GAPDH antibodies were used for confirming the protein loading amount of each sample. The uncropped blots are shown in . ( c ) Three sets of A172 and U343MG cells stably expressing FLAG -tagged WT ROS1 and FLAG -tagged KLC1-ROS1 fusion proteins were established independently, and their cell lysates were analyzed by immunoblotting as shown in b,c. ( d ) Changes in FLAG-tagged WT ROS1, FLAG-tagged KLC1-ROS1 fusion, Akt, ERK1/2, JAK2, and STAT3 activities after expressing FLAG -tagged ROS1 constructs in each cell were quantified by immunoblotting and calculated as follows: (phospho-protein values)/(FLAG-tagged WT ROS1 or FLAG-tagged KLC1-ROS1 fusion values). The mean of the calculated relative activities (value of WT ROS1-expressing cell = 1) is presented as a bar graph. * p > 0.01. N.S.: No Significance.

Article Snippet: Primary antibodies against beta-Actin (#5125), phospho-Akt (Ser 473) (#9271,), total Akt (#9272), c, phospho-Erk 1/2 (Thr 202/Tyr 204) (#9101), total Erk 1/2 (#9102), GAPDH (3683), total JAK2 (#3230), pohspho-JAK2 (Y1007/1008) (#3776), Mcl-1 (#5453), c-Myc (#5605), matrix metalloproteinases 2 (MMP2) (#40994), Poly (ADP-ribose) polymerase (PARP) (#9542), phospho-ROS1 (Y2274/) (#3078), total ROS1 (#3266), phospho-signal transducer and activator of transcription 3 (STAT3, Tyr 705) (#9145), total STAT3 (#9139), alpha-Tubulin (#2125), and HRP-conjugated anti-rabbit secondary antibody (#7074) were purchased from Cell Signaling Technology (Tokyo, Japan).

Techniques: Expressing, Plasmid Preparation, Stable Transfection, Western Blot, Construct

Journal: Cancers

Article Title: KLC1-ROS1 Fusion Exerts Oncogenic Properties of Glioma Cells via Specific Activation of JAK-STAT Pathway

doi: 10.3390/cancers16010009

Figure Lengend Snippet: The KLC1-ROS1 fusion is specifically localized in cytoplasm and associates with JAK2. ( a ) FLAG -tagged wild-type (WT) ROS1 and FLAG -tagged KLC1-ROS1 fusion were stably expressed in A172 cells. After 72 h, the cell lysates were fractionated into plasma membrane, cytosolic, and nuclear fractions. Fractionated cell lysates were analyzed by immunoblotting using the indicated primary antibodies. Primary antibody for e-cadherin, beta actin, and PARP were used as the fraction marker for plasma membrane, cytosol, and nucleus, respectively. # precipitated IgG. ( b ) ( Left ) The cytosolic fractions of cell lysates obtained in a were subjected to immunoprecipitation using anti-FLAG M2 affinity gel, and the precipitates were analyzed by immunoblotting using a primary anti-JAK2 antibody. ( Right ) The input cytosolic fractions were analyzed by immunoblotting using the indicated primary antibodies. Primary anti-beta actin antibody was used for controlling the amount of protein loading in each sample. The uncropped blots are shown in . ( c ) The ratio of JAK2 association efficiency and the ROS1 domain phosphorylation in WT ROS1 and KLC1-ROS1 fusion were calculated from the quantitative results of b. The ratio of JAK2 association efficiency was calculated as follows: (WT ROS1 or KLC1-ROS1 fusion-bound JAK2 value ( b, left ))/(total WT ROS1 or KLC1-ROS1 fusion value ( b, right )). The ratio of ROS1 domain phosphorylation was calculated as follows: (Py-WT ROS1 or KLC1-ROS1 fusion value ( b, right ))/(total WT ROS1 or KLC1-ROS1 fusion value ( b, right )). These ratios were expressed as relative values (WT ROS1 = 1) by the graphs. * p > 0.01.

Article Snippet: Primary antibodies against beta-Actin (#5125), phospho-Akt (Ser 473) (#9271,), total Akt (#9272), c, phospho-Erk 1/2 (Thr 202/Tyr 204) (#9101), total Erk 1/2 (#9102), GAPDH (3683), total JAK2 (#3230), pohspho-JAK2 (Y1007/1008) (#3776), Mcl-1 (#5453), c-Myc (#5605), matrix metalloproteinases 2 (MMP2) (#40994), Poly (ADP-ribose) polymerase (PARP) (#9542), phospho-ROS1 (Y2274/) (#3078), total ROS1 (#3266), phospho-signal transducer and activator of transcription 3 (STAT3, Tyr 705) (#9145), total STAT3 (#9139), alpha-Tubulin (#2125), and HRP-conjugated anti-rabbit secondary antibody (#7074) were purchased from Cell Signaling Technology (Tokyo, Japan).

Techniques: Stable Transfection, Membrane, Western Blot, Marker, Immunoprecipitation

Journal: Cancers

Article Title: KLC1-ROS1 Fusion Exerts Oncogenic Properties of Glioma Cells via Specific Activation of JAK-STAT Pathway

doi: 10.3390/cancers16010009

Figure Lengend Snippet: ROS1–JAK2–STAT3 axis-dependent upregulation of oncogenic properties of KLC1-ROS1 fusion expressed glioma cells compared with wild-type ROS1 expressed cells. ( a ) A172 cells stably expressing FLAG-tagged wild-type (WT) ROS1 or FLAG-tagged KLC1-ROS1 fusion, established in a, were treated with the small-molecule JAK2 inhibitor ruxolitinib (1 μM) or baricitinib (1 μM), as well as vehicle (DMSO; control), as indicated. After 48 h, the total cell number was quantitated, and the cells were subjected to invasion assays. Cell lysates were analyzed using gelatin zymography to detect MMP2 activation. The primary antibody against beta actin was used for confirming the protein loading amount of each sample. * p > 0.01. ( b ) A172 cells stably expressing FLAG-tagged KLC1-ROS1 fusion were treated with the small-molecule ROS1 inhibitor crizotinib (500 nM), lorlatinib (100 nM), or vehicle (control), as indicated. After 48 h, cells were subjected to proliferation and invasion assays. Cell lysates were analyzed using gelatin zymography to detect MMP2 activation. The primary antibody against beta actin was used for controlling the amount of protein loading in each sample. * p > 0.01. The uncropped blots are shown in . ( c ) A172 cells stably expressing FLAG-tagged WT ROS1, FLAG-tagged KLC1-ROS1 fusion, or empty vector, as seen in a, were treated with the small-molecule ROS1 inhibitor lorlatinib (100 nM), the JAK2 inhibitor ruxolitinib (1 μM), and vehicle (DMSO; control), as indicated. After 48 h, the total cell number was quantitated, and the cells were subjected to invasion assays. * p > 0.01. ( d ) The A172 cells used in c were treated with the small-molecule ROS1 inhibitor lorlatinib (100 nM), JAK2 inhibitor ruxolitinib (1 μM), Mcl-1 inhibitor sabutoclax (1 μM), or vehicle (DMSO; control) with vehicle (DMSO) or TMZ (150 μM), as indicated. After 72 h, the cells were subjected to cell death assay. * p > 0.01.

Article Snippet: Primary antibodies against beta-Actin (#5125), phospho-Akt (Ser 473) (#9271,), total Akt (#9272), c, phospho-Erk 1/2 (Thr 202/Tyr 204) (#9101), total Erk 1/2 (#9102), GAPDH (3683), total JAK2 (#3230), pohspho-JAK2 (Y1007/1008) (#3776), Mcl-1 (#5453), c-Myc (#5605), matrix metalloproteinases 2 (MMP2) (#40994), Poly (ADP-ribose) polymerase (PARP) (#9542), phospho-ROS1 (Y2274/) (#3078), total ROS1 (#3266), phospho-signal transducer and activator of transcription 3 (STAT3, Tyr 705) (#9145), total STAT3 (#9139), alpha-Tubulin (#2125), and HRP-conjugated anti-rabbit secondary antibody (#7074) were purchased from Cell Signaling Technology (Tokyo, Japan).

Techniques: Stable Transfection, Expressing, Zymography, Activation Assay, Plasmid Preparation

Journal: Annals of Translational Medicine

Article Title: SOCS3 protein expression predicts the responses of advanced non-small cell lung cancer patients to platinum-based chemotherapy

doi: 10.21037/atm-22-6065

Figure Lengend Snippet: SiRNA-mediated knockdown of SOCS3 (A) enhances JAK2 and STAT3 phosphorylation and the expression of caspase-3 in cultured H1299 and H1437 cells, and is associated with significant increases in the (B) survival rate and (C) drug-induced apoptosis rate of these cells. (D) TUNEL was to assess SOCS3 apoptosis, 200×. *, P<0.05. SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; p, phosphorylated; t, total; JAK2, Janus kinase 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si, small-interfering; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.

Article Snippet: These blots were blocked using 5% non-fat milk before being probed overnight using antibodies specific to SOCS3, phosphorylated JAK2 (p-JAK2), total JAK2 (t-JAK2), phosphorylated STAT3 (p-STAT3), total STAT3 (t-STAT3), caspase-3, or glyceraldehyde-3-phosphate dehydrogenase (all 1:1,000; all from Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Cell Culture, TUNEL Assay, End Labeling

Journal: Annals of Translational Medicine

Article Title: SOCS3 protein expression predicts the responses of advanced non-small cell lung cancer patients to platinum-based chemotherapy

doi: 10.21037/atm-22-6065

Figure Lengend Snippet: JAK2/STAT3 signal pathway mediates SOCS3 to regulate the death of NSCLC cells. (A) The inhibition of JAK2/STAT3 phosphorylation processed with WP1066 (50 mM) dramatically reduced caspase-3 expression in si-SOCS3-transfected lung cancer cells compared to the control cells. WP1066 treatment impaired the pro-survival effects of SOCS3 knockdown in these lung cancer cell lines as evidenced by (B) reductions in the cell survival rate and (C) increases in the cell apoptosis rate. (D) TUNEL was to assess SOCS3 apoptosis, 200×. *, P<0.05. SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; p, phosphorylated; t, total; JAK2, Janus kinase 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide; si, small-interfering; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; NSCLC, non-small cell lung cancer.

Article Snippet: These blots were blocked using 5% non-fat milk before being probed overnight using antibodies specific to SOCS3, phosphorylated JAK2 (p-JAK2), total JAK2 (t-JAK2), phosphorylated STAT3 (p-STAT3), total STAT3 (t-STAT3), caspase-3, or glyceraldehyde-3-phosphate dehydrogenase (all 1:1,000; all from Santa Cruz Biotechnology, CA, USA).

Techniques: Inhibition, Expressing, Transfection, TUNEL Assay, End Labeling