anti phospho jak1  (Cell Signaling Technology Inc)

Journal: CytoJournal

Article Title: Mapping the function of EF-hand domain-containing protein 2 and determining its clinical relevance in non-small-cell lung cancer through single-cell transcriptomics

doi: 10.25259/Cytojournal_29_2025

Figure Lengend Snippet: EFHD2 influences lung cancer cell proliferation, apoptosis, migration, invasion, and various signaling pathways. (a) WB analysis depicting EFHD2 protein expression in normal bronchial epithelial cells (BEAS-2B) and different lung cancer cell lines (A549, NCI-H1299, and HCC827). (b) Relative mRNA levels of EFHD2 in BEAS-2B and lung cancer cell lines, quantified through qRT-PCR. The data are presented as mean ± SD. (c) WB illustrating EFHD2 protein expression levels. (d) qRT-PCR analysis of EFHD2 mRNA expression. (e) CCK-8 assay assessing the impact of EFHD2 OE and KD on cell proliferation; data were presented as OD450 values over 72 h. (f) Flow cytometry analysis evaluating apoptosis rates. (g) Transwell migration and invasion assays. (h) Colony formation assay. (i) WB analysis of JAK and STAT protein expression, using GAPDH as a loading control. Data are expressed as mean ± SD. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001 compared with the A549 group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the NCI-H1299 group. The scale bar represents 100 µm. EFHD2: EF-hand domain-containing protein 2, WB: Western blot, JAK: Janus Kinase, STAT: Signal transducers and activators of transcription, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, qRT-PCR: Quantitative real-time polymerase chain reaction, CCK-8: Cell counting kit 8, OE: Overexpression, KD: Knockdown, mRNA: Messenger RNA, OD450: Optical density at 450 nm, SD: Standard deviation.

Article Snippet: The membranes were then exposed overnight at 4°C to primary antibodies targeting EFHD2 (1:1000, ab24368, Abcam, China), JAK1 (1:1000, HY- P80196 , MedChemExpress, China), phospho-JAK (p-JAK) (1:1000, ab138005, Abcam, China), signal transducers and activators of transcription 3 (STAT3) (1:1000, ab68153, Abcam, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, ab8245, Abcam, China) (loading control).

Techniques: Migration, Protein-Protein interactions, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Colony Assay, Control, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Over Expression, Knockdown, Standard Deviation

Journal: CytoJournal

Article Title: Mapping the function of EF-hand domain-containing protein 2 and determining its clinical relevance in non-small-cell lung cancer through single-cell transcriptomics

doi: 10.25259/Cytojournal_29_2025

Figure Lengend Snippet: EFHD2 regulates cell proliferation, migration, invasion, apoptosis, and JAK/STAT signaling. (a) Co-IP identified that FLAG-EFHD2 interacted with Myc-JAK1 in A549+EFHD2 OE and NCI-H1299+EFHD2 KD cells. (b) CCK-8 detected cell proliferation. Inhibition of JAK decreased the proliferation of A549+EFHD2 OE cells, while activation of JAK increased the proliferation of NCI-H1299+EFHD2 KD cells. (c) Flow cytometry to detect apoptosis. Inhibition of JAK increased the apoptosis of A549+EFHD2 OE cells, whereas JAK agonist enhanced the apoptosis of NCI-H1299+EFHD2 KD cells. (d) Transwell migration and invasion. Inhibition of JAK reduced the migration and invasion of A549+EFHD2 OE cells, whereas activation of JAK promoted the migration and invasion of NCI-H1299+EFHD2 KD cells. (e) Colony formation assay. Inhibition of JAK decreased the colony formation of A549+EFHD2 OE cells, whereas activation of JAK enhanced the colony formation of NCI-H1299+EFHD2 KD cells. (f) Western blotting. JAK inhibition decreased the levels of p-JAK1 and p-STAT3 in A549+EFHD2 OE cells, whereas JAK activation increased the levels of p-JAK1 and p-STAT3 in NCI-H1299+EFHD2 KD cells. Data are expressed as mean ± SD. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001 compared with the A549 group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the NCI-H1299 group. The scale bar represents 100 µm. JAK: Janus Kinase, STAT: Signal transducers and activators of transcription, Co-IP: Co-immunoprecipitation, EFHD2: EF-hand domain-containing protein 2, OE: Overexpression, KD: Knockdown, CCK-8: Cell counting kit 8, p-JAK: Phospho Janus Kinase, p-STAT: Phospho signal transducers and activators of transcription, SD: Standard deviation.

Article Snippet: The membranes were then exposed overnight at 4°C to primary antibodies targeting EFHD2 (1:1000, ab24368, Abcam, China), JAK1 (1:1000, HY- P80196 , MedChemExpress, China), phospho-JAK (p-JAK) (1:1000, ab138005, Abcam, China), signal transducers and activators of transcription 3 (STAT3) (1:1000, ab68153, Abcam, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, ab8245, Abcam, China) (loading control).

Techniques: Migration, Co-Immunoprecipitation Assay, CCK-8 Assay, Inhibition, Activation Assay, Flow Cytometry, Colony Assay, Western Blot, Immunoprecipitation, Over Expression, Knockdown, Cell Counting, Standard Deviation

Journal: bioRxiv

Article Title: Structural Rewiring of IL-7R Dimerization by an Oncogenic Transmembrane Mutation Can Be Reversed by Rational Design

doi: 10.64898/2026.02.17.706319

Figure Lengend Snippet: ( A and B ) Schematic illustrations of BACTH and TOXGREEN analyses, respectively, used for examining homotypic interactions of IL-7R TMD variants. For the BACTH assay, IbaG/IbaG homodimerization was used as the positive control. For the TOXGREEN assay, glycophorin A (GpA), a classical TM dimer, was used as a positive control, while GpA-G83I, a monomeric mutant, served as the negative control. (C) BACTH analysis of TMD interactions for IL-7R TMD variants. Blue colonies indicate interaction between the TMDs in the bacterial inner membrane, while white colonies indicate no such interaction. (D) Quantification of BACTH colony colors normalized relative to the IbaG/IbaG positive control. The data are shown as means ± SEMs calculated from technical replicates (typically three) from three independent experiments. Statistical significance by One-way ANOVA. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (E) TOXGREEN analysis of TMD interactions for IL-7R TMD variants. Quantification of sfGFP activity is normalized relative to the GpA positive control. The data are shown as means ± SEMs calculated from technical replicates (typically five to six) from three independent experiments. Statistical significance by One-way ANOVA. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (F) Phosphorylation of STAT5 (p-STAT5) and JAK1 (p-JAK1) in BaF3 cells expressing WT or mutant IL-7R or co-expressing with γc. The p-STAT5 and p-JAK1 signals were detected by immunoblotting and compared with total STAT5 and JAK1, respectively. ( G-H ) Quantification of p-STAT5 and p-JAK1 signals in (F) as p-STAT5/STAT5 and p-JAK1/JAK1 intensity ratios, normalized to the V253G or V253G/γc. The data are shown as means ± SEMs calculated from three independent experiments. Statistical significance by One-way ANOVA. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: Phospho-STAT5 rabbit mAb (#9359), phospho-Jak1 rabbit mAb (#74129), STAT5 rabbit mAb (#94205), Jak1 rabbit mAb (#3344), and phospho-STAT5 rabbit mAb AF647 (#9365) were purchased from Cell Signaling Technology. β-Actin rabbit mAb (#AC026) and goat anti-rabbit IgG H+L (HRP) (#AS014) were purchased from ABclonal.

Techniques: Positive Control, Mutagenesis, Negative Control, Membrane, Activity Assay, Phospho-proteomics, Expressing, Western Blot

Journal: bioRxiv

Article Title: Structural Rewiring of IL-7R Dimerization by an Oncogenic Transmembrane Mutation Can Be Reversed by Rational Design

doi: 10.64898/2026.02.17.706319

Figure Lengend Snippet: (A) In situ detection of IL-7R V253G dimerization by single-molecule FRET (smFRET) in live cells. IL-7R fused to an N-terminal ALFA-tag was expressed in HeLa cells and labeled in situ with anti-ALFA nanobodies conjugated with donor fluorophore Cy3B or acceptor fluorophore ATTO 643. Dimerization was detected by ALEX-FRET TIRF imaging. (B) Representative smFRET trajectories observed for IL-7R WT and V253G, respectively. Scale bar: 5 µm. (C) Dimerization of IL-7R WT and V253G quantified by smFRET. Data from one representative experiment, with each data point corresponding to the result from one cell. (D) Diffusion constants obtained from IL-7R WT and V253G donor and acceptor channel trajectories (solid circle), and V253G-induced co-trajectories (FRET-channel; open circle). Data from one representative experiment, with each data point corresponding to the result from one of n cells. (E) smFRET efficiency histogram of all co-localized IL-7R V253G pooled from n = 6 cells, and fit by multiple Gaussian functions. The peak with a FRET efficiency of ∼0 (blue fit curve) corresponds to randomly co-localized donor and acceptor molecules. (F) Localization map of co-localized donor and acceptor signals from 150 consecutive frames, color-coded according to the corresponding FRET efficiency. Scale bar: 5 µm. (G) Comparison of dimerization levels for different IL-7RΔECD variants obtained from co-tracking and smFRET detection. Data from three independent experiments, with each data point corresponding to the result from one of n cells. (H) smFRET trajectory length histograms for IL-7RΔECD WT and V253G dimers from one representative experiment pooled from n cells (>1000 trajectories for each condition). (I) Dimerization of IL-7RΔECD V253G in the presence of different JAK1 variants. Data from one representative experiment, with each data point corresponding to the result from one of n cells. Statistical significance in C , D , G and I by Student’s t test. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: Phospho-STAT5 rabbit mAb (#9359), phospho-Jak1 rabbit mAb (#74129), STAT5 rabbit mAb (#94205), Jak1 rabbit mAb (#3344), and phospho-STAT5 rabbit mAb AF647 (#9365) were purchased from Cell Signaling Technology. β-Actin rabbit mAb (#AC026) and goat anti-rabbit IgG H+L (HRP) (#AS014) were purchased from ABclonal.

Techniques: In Situ, Labeling, Imaging, Diffusion-based Assay, Comparison

Journal: bioRxiv

Article Title: Structural Rewiring of IL-7R Dimerization by an Oncogenic Transmembrane Mutation Can Be Reversed by Rational Design

doi: 10.64898/2026.02.17.706319

Figure Lengend Snippet: (A) Schematic illustration of activating homodimer of V253G IL-7R and inactivating homodimer of WT IL-7R. In the V253G IL-7R, residues S249, V253, and I257 form the homodimerization interface, which promotes a JAK1–JAK1 pairing configuration that leads to strong cross phosphorylation. In contrast, the WT IL-7R forms a homodimerization interface composed of F251, L255, I258, and L259, which is incompatible with JAK1-JAK1 cross phosphorylation. (B) TOXGREEN analysis of the impact of single mutations at the L255 face on IL-7R-TMD dimerization. Quantification of sfGFP activity is normalized relative to the GpA positive control. The data are shown as means ± SEMs calculated from technical replicates (typically five to six) from three independent experiments. (C) Effect of disrupting the L255 dimerizing face on ligand-independent p-STAT5 and p-JAK1 signals in BaF3 cells. After 5 h of cytokine deprivation, cells expressing WT or mutant IL-7R (F251A, F258A, F259A) were treated with PBS. The p-STAT5 and p-JAK1 signals were detected by immunoblotting and compared with total STAT5 and JAK1, respectively. ( D-E ) Quantification of p-STAT5 and p-JAK1 signals in (C) as p-STAT5/STAT5 and p-JAK1/JAK1 intensity ratios, normalized to the WT IL-7R. ( F ) Effect of disrupting the G253 dimerizing face on ligand-independent p-STAT5 and p-JAK1 signals in BaF3 cells. After 5 h of cytokine deprivation, cells expressing WT or mutant IL-7R (V253G, V253G/S249Y, V253G/S249A, V257W) were treated with PBS. ( G-H ) Quantification of p-STAT5 and p-JAK1 signals in (F) as p-STAT5/STAT5 and p-JAK1/JAK1 intensity ratios, normalized to the V253G IL-7R. ( I ) Effect of disrupting the G253 dimerizing face on ligand-dependent p-STAT5 and p-JAK1 signals in BaF3 cells. After 5h of cytokine deprivation, cells expressing WT or mutant IL-7R (V253G, V253G/S249Y, V253G/S249A, V257W) were treated with 50 ng/mL IL-7 ( J-K ) Quantification of p-STAT5 and p-JAK1 signals in (I) as p-STAT5/STAT5 and p-JAK1/JAK1 intensity ratios, normalized to the V253G IL-7R.

Article Snippet: Phospho-STAT5 rabbit mAb (#9359), phospho-Jak1 rabbit mAb (#74129), STAT5 rabbit mAb (#94205), Jak1 rabbit mAb (#3344), and phospho-STAT5 rabbit mAb AF647 (#9365) were purchased from Cell Signaling Technology. β-Actin rabbit mAb (#AC026) and goat anti-rabbit IgG H+L (HRP) (#AS014) were purchased from ABclonal.

Techniques: Phospho-proteomics, Activity Assay, Positive Control, Expressing, Mutagenesis, Western Blot