rabbit anti phospho stat5  (Bioss)

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) 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

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 the TMD design concept for competitive blocking of IL-7R V253G homodimerization and ligand-independent receptor activation but not effecting IL-7 induced signaling. (B) Immunoblot analysis of STAT5 phosphorylation (p-STAT5) in stable BaF3 cells expressing the IL-7R V253G mutant and γc, following lentiviral transduction with WT TM PEP or mutants (V253G, L255Y, V253G/L255Y, V253/S249Y). After 12 h of cytokine deprivation, cells were treated with PBS and collected for immunoblotting. (C) Quantification of p-STAT5 signal in (B) as p-STAT5/STAT5 intensity ratios, normalized to the no-delivery control (None). (D) Immunoblot analysis of p-STAT5 in stable BaF3 cells expressing the IL-7R V253G mutant and γc, following lentiviral transduction with WT TM PEP or mutants as in (B). After 12 h of cytokine deprivation, cells were treated with 50 ng/mL IL-7 for 2 h and collected for immunoblotting. (E) Quantification of p-STAT5 signal in (D) as p-STAT5/STAT5 intensity ratios, normalized to the no-delivery control (None). (F) Schematic illustration of the mRNA–LNP delivery system. The mRNA encoding designed TM PEP was delivered into BaF3 cells using lipid nanoparticles (LNPs) comprising ionizable lipids (SM-102) and auxiliary components ( Top ). The mRNAs were modified with N1-methylpseudouridine (m1Ψ) and contained a 5′ Cap1/ARCA and 3′ poly(A) tail (∼100A) ( Middle ). The designed TM PEP sequences were listed with mutated residues in red ( Bottom ). (G) Immunoblot analysis of p-STAT5 in stable BaF3 cells expressing the IL-7R V253G mutant and γc, following mRNA – LNP delivery of WT TM PEP or mutants (V253G, L255Y, V253G/L255Y, V253/S249Y). After 12 h of cytokine deprivation, cells were treated with PBS and collected for immunoblotting. (H) Quantification of p-STAT5 signal in (G) as p-STAT5/STAT5 intensity ratios, normalized to the no-delivery control (None). (I) Immunoblot analysis of p-STAT5 in stable BaF3 cells expressing the IL-7R V253G mutant and γc, following mRNA – LNP delivery of WT TM PEP or mutants as in (G). After 12 h of cytokine deprivation, cells were treated with 50 ng/mL IL-7 for 2 h and collected for immunoblotting. (J) Quantification of p-STAT5 signal in (I) as p-STAT5/STAT5 intensity ratios, normalized to the no-delivery control (None). Unpaired student’s t test was used and data were shown as mean ± SEM calculated from three independent experiments. Represents statistically significant, n.s. (not significant); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. PBS, phosphate-buffered saline. s.e.=short exposure. l.e.= long exposure.

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: Blocking Assay, Activation Assay, Western Blot, Phospho-proteomics, Expressing, Mutagenesis, Transduction, Control, Modification, Saline