jak2 (d2e12) xp® rabbit mab antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: A tumor immune microenvironment gene expression signature for predicting prognosis, immunotherapy efficacy, and drug candidates in triple-negative breast cancer

doi: 10.3389/fimmu.2025.1676768

Figure Lengend Snippet: The immune microenvironment–reprogramming function of NCD is mediated by JAK2 inhibition. (A) Volcano plot of DEGs in MDA-MB-231 cells treated with or without 5 μM NCD. (B) KEGG enrichment analysis of DEGs. (C) Western blot of JAK2, p-JAK2, STAT3, and p-STAT3 in MDA-MB-231 cells treated with increasing doses of NCD (n=3). (D) Quantification of JAK2, p-JAK2, STAT3, and p-STAT3 proteins corresponding to (C) (n = 3). (E) Western blot of JAK2, p-JAK2, STAT3, and p-STAT3 in 4T1 cells treated with increasing doses of NCD (n=3). (F) Quantification of JAK2, p-JAK2, STAT3, and p-STAT3 proteins corresponding to (E) (n = 3). (G) Protein expression levels of JAK2 after knockdown of JAK2 in MDA-MB-231 cell line (n=3). (H) Quantification of JAK2 protein levels corresponding to (G) (n=3). (I) mRNA expression levels of JAK2 , CXCL10 , CXCL11 , EBI3 , FLT3LG after knockdown of JAK2 in MDA-MB-231 cell (n=3). (J) Protein stability of JAK2 under thermal gradient with or without NCD (n=3). (K) CETSA Melting Curve (n=3). (L) SPR sensorgrams of JAK2 binding to NCD. (M) Affinity analysis and KD calculation of JAK2-NCD interaction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Membranes were blocked and then incubated with the following primary antibodies: Stat3 (124H6) Mouse mAb, Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb, Jak2 (D2E12) XP ® Rabbit mAb, Phospho-Jak2 (Tyr1007/1008) Antibody (all from Cell Signaling Technology), and Alpha Tubulin Monoclonal antibody (Proteintech).

Techniques: Inhibition, Western Blot, Expressing, Knockdown, Binding Assay

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: Ba/F3 cells expressing the JAK2-V617F and JAK2-WT were treated with the indicated concentration of the ruxolitinib ( A ) fedratinib ( B ) and lestaurtinib ( C ) for 3 h, and lysates were prepared and subjected to western blotting for the pJAK2, JAK2, pSTAT5 and STAT5. Ba/F3 cells expressing the JAK1-V658F and JAK1-WT were treated for 3.5 h with ruxolitinib, and lysates were analyzed for activation of JAK1 and STAT3 ( D ). Ba/F3 cells stably expressing the CSF3R-T618I and CSF3R-WT treated with indicated concentrations of ruxolitinib ( E ) and G-CSF ( F ) for 3 h, and lysates were subjected to western blotting for the pTYK2, TYK2, pSTAT3, STAT3, pSTAT5 and STAT5. Ba/F3 cells expressing the JAK3-WT were treated with indicated concentrations of ruxolitinib in the presence of interleukin-2 (IL-2), and lysates were subjected to western blotting for the pJAK3, JAK3, pSTAT3 and STAT3 ( G ).

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Expressing, Concentration Assay, Western Blot, Activation Assay, Stable Transfection

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: JAK2-V617F and JAK2-V617F + L983F were stably expressed in Ba/F3 cells and analyzed for ruxolitinib sensitivity with the indicated concentrations. Proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method after incubation for 48 h in the presence of increasing concentrations of the inhibitor ruxolitinib ( A ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. Immunoblot analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentrations of ruxolitinib for 4 h and lysates were subjected to indicated antibodies ( B ). A representative image of n = 2 two independent experiments is shown. JAK2-V617F and JAK2-V617F + L983F were stably expressed in Ba/F3 cells and analyzed for fedratinib sensitivity with the indicated concentrations. Proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method after incubation for 48 h in the presence of increasing concentration of the inhibitor fedratinib ( C ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. Immunoblot analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentrations of fedratinib for 3 h and lysates were subjected to indicated antibodies ( D ). A representative image of n = 2 two independent experiments is shown. Ba/F3 cells expressing JAK1-V658F and JAK1-V658F + L1010F with ruxolitinib and measured the ruxolitinib sensitivity with the indicated concentrations. Proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method after incubation for 48 h in the presence of increasing concentrations of the inhibitor ruxolitinib ( E ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. Immunoblot analysis of Ba/F3 cells expressing JAK1 mutants cultured with indicated concentrations of ruxolitinib for 3 h and lysates were subjected to indicated antibodies ( F ). A representative image of n = 2 two independent experiments is shown. Similarly, Ba/F3 cells expressing the JAK1 variants with the indicated concentration of fedratinib and measured the fedratinib sensitivity with the indicated concentrations. Proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method after incubation for 48 h in the presence of increasing concentration of the inhibitor fedratinib ( G ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. Immunoblot analysis of Ba/F3 cells expressing JAK1 mutants cultured with increasing concentrations of fedratinib (0, 250, 500, 1000, 2000, 4000 and 8000 nM) for 4 h and lysates were subjected to indicated antibodies ( H ). A representative image of n = 2 two independent experiments is shown. **** p < 0.0001; ** p < 0.01, * p < 0.05 and n.s., not significant, p > 0.05 by one-way ANOVA test.

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Stable Transfection, Incubation, Standard Deviation, Western Blot, Expressing, Cell Culture, Concentration Assay

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: JAK2-V617F expressing Ba/F3 cells were treated without and with vanadate, ruxolitinib and fedratinib. Cells were lysed in a non-denaturing lysis buffer, and immunoprecipitation was carried out using the anti-flag antibody, anti-JAK2 antibody and phosphospecific Tyr1007/Tyr1008 JAK2 antibody. Immunocomplexes and whole-cell lysate (WCL) were analyzed by IB ( A ). Denaturing immunoprecipitation was carried out using the same antibodies mentioned in non-denaturing immunoprecipitation, and these results showed all the antibodies were able to immunoprecipitate JAK2 equally either in the presence or in the absence of ATP-competitive inhibitors ( B ). A representative image of n = 2 two independent experiments is shown.

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Expressing, Lysis, Immunoprecipitation

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: HEK293Tcells expressing the JAK2-V617F, JAK2-V617F + R975A and JAK2-V617F + K999A were serum starved and treated with 1 μM pervanadate and without and lysates were subjected with indicated antibodies ( A ). The proliferation of parental Ba/F3 cells and Ba/F3 cells expressing JAK2-V617FJAK2, JAK2-V617F + R975A and JAK2-V617F + K999A in the absence of interleukin-3 (IL-3) was quantified by the relative optical density (OD) after 96 h using MTS-based assay ( B ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. JAK2-V617F and JAK2-V617F + R975A were stably expressed in Ba/F3 cells and analyzed for ruxolitinib sensitivity with the indicated concentrations. Proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method after incubation for 48 h in the presence of increasing concentrations of the inhibitor ruxolitinib ( C ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. Immunoblot analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentrations of ruxolitinib for 4 h and lysates were subjected to indicated antibodies ( D ). A representative image of n = 2 two independent experiments is shown. JAK2-V617F and JAK2-V617F + K999A were stably expressed in Ba/F3 cells and analyzed for ruxolitinib sensitivity with the indicated concentrations. Proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method after incubation for 48 h in the presence of increasing concentration of the inhibitor ruxolitinib ( E ). Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density. Immunoblot analysis of Ba/F3 cells expressing JAK2 mutants cultured with increasing concentrations of ruxolitinib (0, 100, 200, 400, 800, and 1000 nM) for 4 h, and lysates were subjected to indicated antibodies ( F ). A representative image of n = 2 two independent experiments is shown. **** p < 0.0001; ** p < 0.01, * p < 0.05 and n.s., not significant, p > 0.05 by one-way ANOVA test.

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Expressing, MTS Assay, Standard Deviation, Stable Transfection, Incubation, Western Blot, Cell Culture, Concentration Assay

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: Ba/F cells expressing JAK2-V617F cells were treated for one hour with ruxolitinib and DMSO, and after one hour of pretreatment, ruxolitinib and DMSO is washed out from the cells with PBS (3 times) and incubated in the absence of inhibitor with indicated time points and lysates were measured for STAT5, AKT and ERK activity ( A ). Quantification of phsopho-STAT5 was measured with indicated time periods when lysates were prepared from ruxolitinib wash and DMSO wash ( B ). Similarly, ruxolitinib washed out and DMSO washed out samples were incubated with the indicated time period to measure the c-Myc, PIM1and Bcl-2 levels ( C ). Ba/F3 cells expressing the JAK2-V617F were treated with 1 μM ruxolitinib or DMSO for a time period of 45 min, and ruxolitinib and DMSO were washed out from cells and incubated for a period of 24-h and measured the cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (MTS)- based method. Data is shown as mean ± standard deviation (SD) ( n = 3). OD—optical density ( D ). ** p < 0.01, * p < 0.05 and n.s., not significant, p > 0.05 by Student’s t test.

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Expressing, Incubation, Activity Assay, Standard Deviation

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: The proteomap illustrates the most altered kinases, represented as polygon-shaped tiles, with their mean kinase statistics. Proteins within the same category are color-coded similarly and positioned adjacently to form larger regions. In this case, the activity of all displayed kinases is reduced compared to the control (DMSO) due to the effective inhibition of JAK kinases by ruxolitinib. The size of each polygon is proportional to the magnitude of change ( A ). Tyrosine and serine/threonine kinases affected by ruxolitinib inhibition of JAK1/2 in the phylogenetic kinome tree ( B ). String network (medium confidence of 0.400) for kinases influenced by the ruxolitinib inhibition of JAK1/2 (JAK1/2 shown in red, blue = reduction of kinase activity), represented by the mean kinase statistic ( C ). PIM1 and PIM2 were ectopically expressed in Ba/F3 JAK2-V617F cells, and lysates were subjected to western blotting with the indicated antibodies ( D ). PIM1 and PIM2 were stably knockdown in Ba/F3 JAK2-V617F cells, and lysates were subjected to western blotting with the indicated antibodies ( E ). Single clones of Ba/F3 cells expressing JAK2-V617F with PIM1 and PIM2 kinases grown in 96-well plates in the presence of 4 μM ruxolitinib were counted. The number of resistant clones per million cells input is shown ( F ). Single clones of Ba/F3 JAK2-V617F with PIM1 and PIM2 knockdown cells in 96-well plates with 4 μM ruxolitinib were counted. The number of resistant clones per million cells input is shown ( G ). ** p < 0.01, * p < 0.05 and n.s., not significant, p > 0.05 by Student’s t test.

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Activity Assay, Control, Inhibition, Western Blot, Stable Transfection, Knockdown, Clone Assay, Expressing

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: In the presence of ATP, activation loop Tyr1007/Tyr1008 of JAK2-V617F is phosphorylated, which leads to activation of STAT5 and PIM-kinases ( A ). Ruxolitinib binds to the kinase domain of JAK2, when activation loop Tyr1007/Tyr1008 are fully phosphorylated. This binding stabilizes the activation loop conformation inside the kinase domain of JAK2. Thus, the phosphatases cannot access the phosphorylated tyrosine residues of activation loop, however JAK2 in this conformation exists in an inhibited state ( B ). Arg975 and Lys999 residues help in the stabilization of the activation loop conformation inside the kinase domain ( C ). When hyperphosphorylated JAK2 is dissociated from ruxolitinib, it leads to hyperactivation of STAT5 and PIM-kinases ( D ).

Article Snippet: JAK2 c-terminal antibody (D2E12 XP R ), pSTAT5, pTYK2, TYK2, pJAK3, JAK3, pAkt, pERK, ERK, c-Myc, BCL-2, Akt were purchased from cell signaling.

Techniques: Activation Assay, Binding Assay