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

Journal:

Article Title: Prolactin and ErbB4/HER4 Signaling Interact via Janus Kinase 2 to Induce Mammary Epithelial Cell Gene Expression Differentiation

doi: 10.1210/me.2008-0055

Figure Lengend Snippet: ErbB4 Is Required for JAK2 Activation in Response to HB-EGF But Not PRL

Article Snippet: Western Analysis and Immunoprecipitation Cells were lysed, and proteins were precipitated with the following antibodies as described previously ( 57 ): rabbit polyclonal anti-ErbB1 and -ErbB4 (C terminus) generated in this laboratory and previously described ( 57 ); JAK2, PRLR, phosphotyrosine (PY20), Src, and P-Src (Santa Cruz Biotechnologies, Santa Cruz, CA), STAT5A and P-Tyr 694 STAT5 (Zymed Laboratories, San Francisco, CA), p44/42, and phospho-p44/42 (Cell Signaling Technologies, Beverly, MA).

Techniques: Activation Assay

Journal:

Article Title: Prolactin and ErbB4/HER4 Signaling Interact via Janus Kinase 2 to Induce Mammary Epithelial Cell Gene Expression Differentiation

doi: 10.1210/me.2008-0055

Figure Lengend Snippet: Kinase Activity and Expression of JAK2 Is Required for ErbB4-Mediated STAT5A Activation

Article Snippet: Western Analysis and Immunoprecipitation Cells were lysed, and proteins were precipitated with the following antibodies as described previously ( 57 ): rabbit polyclonal anti-ErbB1 and -ErbB4 (C terminus) generated in this laboratory and previously described ( 57 ); JAK2, PRLR, phosphotyrosine (PY20), Src, and P-Src (Santa Cruz Biotechnologies, Santa Cruz, CA), STAT5A and P-Tyr 694 STAT5 (Zymed Laboratories, San Francisco, CA), p44/42, and phospho-p44/42 (Cell Signaling Technologies, Beverly, MA).

Techniques: Activity Assay, Expressing, Activation Assay

Journal: Oncogene

Article Title: EGFR-Stat3 signalling in nerve glial cells modifies neurofibroma initiation

doi: 10.1038/onc.2016.386

Figure Lengend Snippet: (a) Western blot of mouse sciatic nerves that are WT, EGFR-overexpressing (EGFR/EGFR), or with diminished levels of Egfr activity (Wa2), showing that Stat3 activity is increased in EGFR/EGFR nerves. (b) Western blot of mouse neurofibroma-derived EGFR+ progenitor-like sphere cells OSI-774. Inhibition of EGFR function inhibits Stat3-Y705 phosphorylation (P-Stat3). Total Stat3 (Stat3) was used as loading control. Statistics B, C, E: unpaired t test, two-tailed. (c–d) Immunofluorescent staining showing that P-Stat3 is detected in Nf1fl/fl;DhhCre mouse neurofibromas (C, CTRL) and decreased in Nf1fl/fl;DhhCre,Wa2 neurofibroma (D, Wa2). Bar = 20 µm. (e) Quantification of P-Stat3+ cells in Nf1fl/fl;DhhCre (white bar) and Nf1fl/fl;DhhCre,Wa2 (black bar). (f) Western blot of neurofibroma lysates from Nf1fl/fl;DhhCre and Nf1fl/fl;DhhCre;Wa2 mice. (g) Western blot of lysates from Nf1fl/fl;DhhCre mouse neurofibromas treated with AZD1480 or vehicle for 5 days. (f and g) Stat3 and anti-β-actin serve as loading controls. Antibodies: P-Stat3, Cell Signaling, #9145; Stat3, Cell Signaling, #4904; EGFR, Santa Cruz, #SC-03; P-EGFR, Santa Cruz, #SC-12351; P-Jak2, Cell Signaling, # 3776; Jak2, Cell Signaling, #3230; β-actin, Cell Signaling, #5125. ***P <0.001.

Article Snippet: Antibodies: P-Stat3, Cell Signaling, #9145; Stat3, Cell Signaling, #4904; EGFR, Santa Cruz, #SC-03; P-EGFR, Santa Cruz, #SC-12351; P-Jak2, Cell Signaling, # 3776; Jak2, Cell Signaling, #3230; β-actin, Cell Signaling, #5125.

Techniques: Western Blot, Activity Assay, Derivative Assay, Inhibition, Two Tailed Test, Staining

Journal: Oncogene

Article Title: EGFR-Stat3 signalling in nerve glial cells modifies neurofibroma initiation

doi: 10.1038/onc.2016.386

Figure Lengend Snippet: (a) A representative western blot of WT mouse sciatic nerve and Nf1fl/fl;DhhCre mouse neurofibroma lysates showing expression of II6 protein (II-6, Abcam, Cambridge, MA, USA; #AB6672). Anti-β-actin served as a loading control. (b) II-6 quantification on OSI-774, FLLL32, and DMSO treated medium conditioned by mouse neurofibroma spheres. Sphere medium without treatment was also used as additional control (n = 3 for each group). Unpaired t test, two-tailed was used. (c) Mouse neurofibroma sphere counts on II-6 antibody (II-6 Ab), OSI-774, II-6 Ab+OSI-774, IgG+DMSO treated mouse neurofibroma spheres. (d) Western blot of P-Jak2, Jak2, P-Stat3 on II-6 antibody (II-6 Ab), OSI-774, II-6 Ab+OSI-774, IgG+DMSO treated mouse neurofibroma spheres. Anti-Stat3 and anti-β-actin serve as controls. (e) Sphere counts show that two shII-6 shRNAs (#1 and #2) each significantly decrease mouse neurofibroma sphere formation, compared to non-target lentivirus yellow fluorescent protein control. (f) Western blot showing knockdown of II-6 in Nf1fl/fl;DhhCre mouse neurofibroma spheres, 4 days after sh II-6 infection using two different shRNA clones (#1, #2). Numbers below indicate the relative band intensity (50% and 40%), normalized to β-actin for each sample. (g) Inhibitory effects of the JAK1/2 inhibitor AZD1480 on Nf1fl/fl;DhhCre, and Nf1fl/fl;DhhCre;EGFR but not Nf1fl/fl;DhhCre;Wa2 mouse neurofibroma spheres. DMSO was used as control. The JAK3 inhibitor CP 690550 did not affect sphere number at concentrations below 1 µM. Three independent experiments were performed, and data are represented as mean ± s.e.m. Statistics: B, one-way ANOVA; E, unpaired t test, two-tailed. *P <0.05, **P <0.01, ***P <0.001.

Article Snippet: Antibodies: P-Stat3, Cell Signaling, #9145; Stat3, Cell Signaling, #4904; EGFR, Santa Cruz, #SC-03; P-EGFR, Santa Cruz, #SC-12351; P-Jak2, Cell Signaling, # 3776; Jak2, Cell Signaling, #3230; β-actin, Cell Signaling, #5125.

Techniques: Western Blot, Expressing, Two Tailed Test, Infection, shRNA, Clone Assay

Journal: Scientific Reports

Article Title: LncTUG1 promotes hepatocellular carcinoma immune evasion via upregulating PD-L1 expression

doi: 10.1038/s41598-023-42948-8

Figure Lengend Snippet: TUG1 affects the JAK2/ STAT3 pathway to regulate PD-L1. ( A ): The relative mRNA expression of JAK2 was analyzed in shTUG1 HCC cells. ( B ): The relative mRNA expression of STAT3 was analyzed in shTUG1 HCC cells. ( C ): The protein levels of pJAK2, JAK2, pSTAT3, STAT3, PD-L1 were analyzed in shTUG1 HCC cells by western blotting. ( D ) pJAK2, JAK2,and PDL1 protein levels are assessed by immunoblotting in HCC-LM3 cell was transfected with TUG1 silenced (shTUG1) and/or JAK2 overexpressed (JAK2-OE). ( E ) pSTAT3,STAT3,and PDL1 protein levels are assessed by immunoblotting in HCC-LM3 cell was transfected with TUG1 silenced (shTUG1) and/or STAT3 overexpressed (STAT3-OE).

Article Snippet: Protein detection was performed by using the following primary antibodies: anti-PD-L1 (ab205921, Abcam), anti-GAPDH (ab8245, Abcam), anti-JAK2 (#3230, Cell Signaling Technology), anti-pJAK2 Tyr1007/1008 (#3771,Cell Signaling Technology),anti-STAT3 (#9139, Cell Signaling Technology), anti-pSTAT3 Tyr705 (#9145, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Transfection

Journal: Oncology Letters

Article Title: Ganoderic acid A exerts antitumor activity against MDA-MB-231 human breast cancer cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

doi: 10.3892/ol.2018.9475

Figure Lengend Snippet: GA-A affected JAK2/STAT3 signaling pathway and mitochondrial apoptotic pathway associated proteins in MDA-MB-231 cells. (A) The bands of western blot analysis. β-actin was used as an internal control. (B) Statistical charts of the relative expression levels of t-JAK2 and p-JAK2. (C) Statistical charts of the relative expression levels of t-STAT3 and p-STAT3. (D) Statistical charts of the relative expression levels of Bcl-xL. (E) Statistical charts of the relative expression levels of Mcl-1. (F) Statistical charts of the relative expression levels of Bax. (G) Statistical charts of the relative expression levels of Bak. (H) Statistical charts of the relative expression levels of cytosolic Cytochrome c. ***P<0.001, **P<0.01, *P<0.05 vs. GA-A-untreated control. GA-A, Ganoderic acid A; STAT, signal transducer and activator of transcription; JAK, Janus kinase; Bcl-xL, B cell lymphoma-extra-large; Mcl-1, myeloid cell leukemia 1; Bax, Bcl2 associated X protein; Bak, Bcl2 antagonist/killer; n.s., not significant; p-, phosphorylated; t-, total.

Article Snippet: JAK2, p-JAK2, p-STAT3, STAT3 primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: Oncology Letters

Article Title: Ganoderic acid A exerts antitumor activity against MDA-MB-231 human breast cancer cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

doi: 10.3892/ol.2018.9475

Figure Lengend Snippet: Effects of GA-A combined with AG490 on JAK2/STAT3 signaling. (A) The bands of western blot analysis. β-actin was used as an internal control. Relative expression levels of (B) p-JAK2 and (C) p-STAT3. ***P<0.001, **P<0.01, *P<0.05 vs. GA-A-untreated control. GA-A, Ganoderic acid A; STAT, signal transducer and activator of transcription; JAK, Janus kinase; n.s., not significant; p-, phosphorylated; t-, total.

Article Snippet: JAK2, p-JAK2, p-STAT3, STAT3 primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

doi: 10.1186/s13046-019-1277-x

Figure Lengend Snippet: High EREG expression promotes NF-CAF transition through the JAK2-STAT3 pathway. a & b , Representative images and quantitative analysis of western blotting showing that EREG overexpression in NFs and EREG interference in CAFs were successful. Moreover, the expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression and downregulated after EREG interference. GAPDH was used as a loading control. c & d & e , Both phosphorylated forms of JAK2 and STAT3 and the p-JAK2/JAK2 and p-STAT3/STAT3 ratios were significantly augmented after EREG overexpression. The change in expression of major participants of other pathways after EREG overexpression was not as significant. GAPDH was used as a loading control. f & g & h , Representative images and quantification of western blotting showing that expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression but was reduced after treatment with the JAK2 inhibitor AG490, indicating that AG490 antagonizes EREG-mediated NF activation. CAF markers, as well as (p-)JAK2 and (p-)STAT3 expression and p-JAK2/JAK2 and p-STAT3/STAT3 ratios decreased after EREG interference in CAFs but were restored by IL-6 treatment. GAPDH was used as a loading control. i , ELISA showing the IL-6 level in the CM of NFs/CAFs after the indicated treatment. J&K, Representative images and quantification of immunohistochemical staining in clinical samples revealed that high EREG expression was correlated with high phospho-JAK2 and phospho-STAT3 expression, while low EREG expression was correlated with low phospho-JAK2 and phospho-STAT3 expression. Magnification: 200×. *: p < 0.05, **: p < 0.01, ***: p < 0.001

Article Snippet: Western blots were performed using an SDS–PAGE electrophoresis system as described previously [ ], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies.

Techniques: Expressing, Western Blot, Over Expression, Control, Activation Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

doi: 10.1186/s13046-019-1277-x

Figure Lengend Snippet: High EREG expression in fibroblasts promotes tumor growth in vivo. a , Photographs of tumor formation in nude mice and tumor xenografts 4 weeks after inoculation. b & c , Tumor growth curves measured after injection of HSC3 cells or conditioned fibroblasts as indicated. The tumor volume was calculated every 7 days until termination. d - g , RT-PCR analysis of Ereg, Jak2, Stat3 and Il6 expression in tissues of resected tumors, revealing successful overexpression/interference of Ereg and associated upregulation or downregulation of Jak2, Stat3 and Il6 . h , Immunohistochemical staining showed that tumors that developed from EREG-overexpressing NFs had a higher level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors that developed from control NFs. Tumors that developed from siEREG-transfected CAFs showed a lower level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors developed from siNC-transfected CAFs. S: stroma, T: tumor. Magnification: 200×. i & j , Western blotting showed that CAFs significantly promote EMT in vivo compared with NFs, with decreased E-cadherin expression and increased N-cadherin and vimentin expression. This EMT-promoting ability was attenuated after EREG knockdown. On the other hand, EREG overexpression in NFs gave NFs a CAF-like EMT-promoting ability. k , Increased EREG expression in NFs led to acquisition of the CAF phenotype in a JAK2-STAT3-dependent way and supported OSCC invasion through the promotion of EMT in tumor cells

Article Snippet: Western blots were performed using an SDS–PAGE electrophoresis system as described previously [ ], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies.

Techniques: Expressing, In Vivo, Injection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Immunohistochemical staining, Staining, Control, Transfection, Western Blot, Knockdown

Journal: American Journal of Translational Research

Article Title: Novel apoE receptor mimetics reduce LPS-induced microglial inflammation

doi:

Figure Lengend Snippet: 6KApoEp attenuates LPS-induced JAK2 and STAT3 phosphorylation. BV2 cells were treated with LPS at 100 ng/ml in the absence or presence of 6KApoEp at 10 µM for 0, 15, 30 or 60 min. Total (JAK2 and STAT3) and phosphorylated JAK2 and STAT3 (p-JAK2 and p-STAT3) in cell lysates was then determined by WB, utilizing anti-total JAK2 (Cell Signaling, #3230), anti-total STAT3 (#9132), anti-phospho-JAK2 (#3774) and anti-phospho STAT3 antibodies (#9131). Representative blots are shown for each condition and band density ratios of p-JAK2 to JAK2 and p-STAT3 to STAT3 determined by densitometry analysis are shown below each blot. Full non-adjusted images of WB shown in Figure S2. LPS-induced JAK2 and STAT3 phosphorylation was reduced by 6KApoEp between 15 and 60 min of treatment. Total JAK2 and STAT3 levels were similar across treatment conditions. Results are expressed as mean ± S.E.M. Asterisk indicate P < 0.05 compared with LPS alone.

Article Snippet: BV2 cells were treated with LPS at 100 ng/ml in the absence or presence of 6KApoEp at 10 µM for 0, 15, 30 or 60 min. Total (JAK2 and STAT3) and phosphorylated JAK2 and STAT3 (p-JAK2 and p-STAT3) in cell lysates was then determined by WB, utilizing anti-total JAK2 (Cell Signaling, #3230), anti-total STAT3 (#9132), anti-phospho-JAK2 (#3774) and anti-phospho STAT3 antibodies (#9131).

Techniques: