pstat5  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Engineered probiotics for tumor-targeted combination chemoimmunotherapy

doi: 10.64898/2026.02.04.703875

Figure Lengend Snippet: (A) Schematic illustration showing the co-expression of pCD, PD-L1 nanobody (PDL1nb) and IL-15Rα sushi -IL-15 (s15) fusion protein in EcNx ΔpreTA bacteria. (B) Western blot image showing the expression of pCD by EcNx ΔpreTA -pCD and EcNx ΔpreTA -pCD/PDL1nb/s15 strains. The dashed black line indicates the cropping to remove a non-relevant lane. (C) Merged flow cytometric histogram (left) and percentage of PD-L1 + MC38 cells (right) showing the production and activity of PDL1nb. MC38 cells were pre-incubated with the lysates of indicated strains for 20 min and then stained with APC-labeled PD-L1 antibody for flow cytometric analysis. LB, lysogeny broth. (D) ELISA quantification of IL-15Rα sushi -IL-15 concentration in the lysates of EcNx ΔpreTA , EcNx-s15 and EcNx ΔpreTA -pCD/PDL1nb/s15 bacteria. (E) Western blot detection of phosphorylated STAT5 (pSTAT5) and STAT5. Freshly isolated splenocytes were stimulated with indicated bacteria lysates for 30 min and then collected for western blot. (C and D) Data are presented as mean ± sem. (C) ns, not significant; ** p < 0.01; **** p < 0.0001; One-way ANOVA with Fisher’s LSD test.

Article Snippet: Cells were then collected by centrifugation (450 g , 5 min), and boiled in 1× SDS loading dye for WB detection of pSTAT5 (Cell Signaling, Phospho-Stat5 (Tyr694) Antibody #9351).

Techniques: Expressing, Bacteria, Western Blot, Activity Assay, Incubation, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Concentration Assay, Isolation

Journal: Life Science Alliance

Article Title: TRPM7 and magnesium orchestrate human CD4 T-cell activation and differentiation

doi: 10.26508/lsa.202503357

Figure Lengend Snippet: (A) Representative Western blot of total AKT1 signals in total CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO (Ctrl) in resting state and after 30 min α-CD3/α-CD28 stimulation. (B) Respective quantification of total AKT1 signals from blots shown in (A), n = 5. (C) Representative Western blot of total SMAD2/3 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min TGF-β stimulation. (D) Respective quantification of total SMAD2/3 signals from blots shown in (D), n = 4. (E) Representative Western blot of total SMAD2/3 signals in CD4 T cells in presence of 6 mM MgCl 2 (MgCl 2 ) or H 2 O control (Ctrl) in resting state and after 10 min TGF-β stimulation. (F) Respective quantification of total SMAD2/3 signals from blots shown in E, n = 4. (G) Representative Western blot of pSTAT3 Tyr705 signals in CD4 T cells, in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl), shown in resting state and after 10 min stimulation with IL-6. (H) Respective quantification of pSTAT3 Tyr705 signals of blots shown in (G). Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 3. (I) Representative FACS plots of pSTAT3 Tyr705 signal in CD4 T cells upon 10 min stimulation with IL-6. Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated Ctrl shown in light gray. (J) Respective quantification of pSTAT3 Tyr705 signal in CD4 T cells of data shown in (I), n = 4. (K) Representative FACS plots of pSTAT3 Tyr705 signal in CD4 T cells upon 10 min stimulation with a-CD3/a-CD28 for cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (L) Respective quantification of pSTAT3 Tyr705 signal of data shown in (K), n = 4. (M) Representative Western blot of pSTAT3 Ser727 signals in CD4 T cells in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min stimulation with IL-6. (N) Respective quantification of pSTAT3 Ser727 signals of blots shown in (M). Cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), n = 3. (O) Representative FACS plots of pSTAT3 Ser727 signal in CD4 T cells upon 10 min stimulation with IL-6 of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (P) Respective quantification of pSTAT3 Ser727 signal of data shown in (O), n = 4. (Q) Representative Western blot of pSTAT3 Ser727 signals in CD4 T cells, in presence of 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 10 min stimulation with a-CD3/a-CD28. (R) Respective quantification of pSTAT3 Ser727 signals of blots shown in (Q). (S) Representative FACS plots of pSTAT3 Ser727 signal in CD4 T cells upon 10 min stimulation with a-CD3/a-CD28 of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (T) Respective quantification of pSTAT3 Ser727 signal of data shown in (S), n = 4. (U) Representative Western blot of pSTAT5 Tyr694 signals in CD4 T cells of cells treated with 30 μM NS8593 (NS) or DMSO control (Ctrl) in resting state and after 15 min a-CD3/a-CD28 stimulation. (V) Representative FACS plot of pSTAT5 Tyr694 signal in CD4 T cells upon 15 min stimulation with IL-2 of cells treated with 30 μM NS8593 (NS, red) or DMSO control (Ctrl, black), unstimulated control in light gray. (W) Respective quantification of pSTAT5 Tyr694 signal of data shown in (V), n = 4. (A, D, F, H, J, L, N, P, R, T, W) Statistics: One-way ANOVA (A, D, F, H, N, R) and t test (J, L, P, T, W). *** P < 0.0005, **** P < 0.0001 and n.s., not significant. Data are mean ± SD.

Article Snippet: The following antibodies were used: α-pSMAD2 Ser465/Ser467 (138D4; Cell Signaling), α-SMAD2/3 (D7G7; Cell Signaling), α-AKT1 (D9-9-C9; Thermo Fisher Scientific), α-pSTAT3 Tyr705 (D3A7; Cell Signaling), α-pSTAT3 Ser727 (D8C2Z; Cell Signaling), α-pSTAT5 Tyr 694 (D47E7; Cell Signaling), α-GAPDH (G-9; Santa Cruz).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Prolactin Shapes Cortical Plasticity in Fathers

doi: 10.1101/2025.10.20.683400

Figure Lengend Snippet: (a) Representative fluorescent micrographs of pSTAT5 immunostaining in ACx in three experimental groups. Top: low resolution coronal slice. Bottom: high magnification images of the ACx (marked with a white triangle in the top). (b–c) Quantification of pSTAT5+ signal in ACx across the three groups (b). Layer specific pSTAT5+ signal in ACx in post-mated males and fathers. p-values (b). One-way ANOVA: 2.876e-11. PM vs F: 9.6e-10, F vs PF: 2.7e-8, PM vs PF 0.42, after Tukey-Kramer multiple comparison correction. Data were collected from 12 brain slices per mouse, across 5 mice. LME corrected p values (c): L1:3.4e-21, L2-3: 7.9e-20, L4: 3.7e-18, L5-6: 8.7e-17. (d) Level of prolactin in postmated males and fathers (n = 8 and 8 mice, for both post-mating and father time points. p = 0.66, Unpaired t-test). (e) Relative expression levels of the long isoform of prolactin receptor (Prlr-L) in ACx of fathers (n = 3 and 3 mice, for both post-mating and father time points. p = 0.0028, Unpaired t-test). Data are median ± IQR; *p<0.05, **p<0.01, **p<0.005 by unpaired t-test or One-way ANOVA followed by Tukey-Kramer correction for multiple comparisons or LME followed by FDR for multi comparison correction.

Article Snippet: After PBS wash, sections were incubated in rabbit pSTAT5 primary antibody (pSTAT5 Tyr 694, Cat#: C11C5, 1:500; Cell Signaling Technology) for 72 hours at 4°C, followed by two-hours incubation in goat anti-rabbit Cy5 conjugated secondary antibody (1:250; Jackson ImmunoResearch, Cat #111-175-144, RRID: AB_2338013).

Techniques: Immunostaining, Comparison, Expressing

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( a ) Representative fast FLIM images of HEK-Blue TM IL-2 cells transfected with STATeLight5A 30 min after stimulation with titrated concentrations of IL-2. Fluorescence lifetime is represented by a color-coded scale. Scale bars, 10 µm. ( b ) Percentage of pSTAT5 + HEK-Blue TM IL-2 cells treated with varying IL-2 concentrations, as assessed flow cytometry. ( c ) Correlation between mNG fluorescence lifetime and pSTAT5 + cells. Statistical significance was tested with Pearson correlation coefficient, R = 0.91, *** p = 0.0003. ( d ) NativePAGE-western blot showing STAT5 dimerization at different IL-2 concentrations. ( e ) HEK-Blue TM reporter assay showing STAT5 transcriptional activation at different IL-2 concentrations. Data are shown as mean ± SEM, n = 3 biological replicates. ( f ) Correlation between the dimer/monomer ratio, assessed by western blot and STAT5 transcriptional activation, assessed by HEK-Blue TM reporter assay. Statistical significance was tested with Pearson correlation coefficient, R = 0.98, **** p = 9.17 × 10 −6 . ( g ) Correlation between fluorescence lifetime and STAT5 transcriptional activation, assessed by HEK-Blue TM reporter assay. Statistical significance was tested with Pearson correlation coefficient, R = -0.98, *** p = 0.0002.

Article Snippet: The membrane was blocked with 5% skim milk in 0.1% Tween-20 in Tris-buffered saline pH 7.4 (TBS-T) for 2 h, followed by incubation with primary antibodies overnight and subsequently secondary anti-horseradish peroxidase (HRP) antibodies for 2 h. Primary antibodies including anti-pSTAT5 (Y694) antibody (Cell Signaling, 9351; 1:3,000 dilution), anti-STAT5A monoclonal antibody (TrueMAB, Thermo Fisher Scientific, clone OTI4E7; 1:3,000 dilution) and anti-actin antibody (Merck, MAB1501R, clone C4; 1:1,000 dilution) and secondary antibodies including anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074; 1:4,000 dilution) and goat anti-mouse HRP-conjugated antibody (Bethyl, A90-116P; 1:2,000 dilution) were diluted in 5% skim milk in TBS-T before use.

Techniques: Transfection, Fluorescence, Flow Cytometry, Western Blot, Reporter Assay, Activation Assay

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( a ) Representative fast FLIM images of HEK-Blue TM IL-2 cells transfected with STATeLight5A and stimulated with 3.5 µg of IL-2 over a time course of 90 minutes. Scale bars, 10 µm. ( b ) Immunoprecipitation coupled with western blot detecting endogenous STAT5B which interacts with STAT5-mSC-I-FLAG in HEK-Blue TM IL-2. ( c ) Immunoprecipitation coupled with western blot detecting endogenous STAT5B which interacts with STAT5-mNG-I-Myc in HEK-Blue TM IL-2. ( d ) Representative flow cytometry plots showing endogenous pSTAT5 time dynamics in IL-2–stimulated HEK-Blue TM IL-2 cells expressing either STATeLight5A (gated on mNG⁺mSC-I⁺ cells) or wild-type (WT) STAT5A-Myc (gated on Myc⁺ cells). ( e ) Quantification of pSTAT5 levels shown in ( d ). ( f ) HEK-Blue TM IL-2 cells were transduced with either scrambled short-hairpin RNA (scr shRNA) or STAT5B shRNA. STAT5B abundance after knock-down was assessed by western blot using anti-STAT5B antibodies. ( g ) Quantification of mNG fluorescence lifetime in untreated cells (n = 27), scr shRNA-expressing cells (n = 26), or STAT5B knockdown cells (n = 15), either in unstimulated or IL-2-stimulated conditions. Boxplots show the median (center line), 25th and 75th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 1.21 × 10 −14 (Untransduced), **** p = 1.21 × 10 −14 (scr shRNA), **** p = 1.29 × 10 −8 (STAT5B shRNA).

Article Snippet: The membrane was blocked with 5% skim milk in 0.1% Tween-20 in Tris-buffered saline pH 7.4 (TBS-T) for 2 h, followed by incubation with primary antibodies overnight and subsequently secondary anti-horseradish peroxidase (HRP) antibodies for 2 h. Primary antibodies including anti-pSTAT5 (Y694) antibody (Cell Signaling, 9351; 1:3,000 dilution), anti-STAT5A monoclonal antibody (TrueMAB, Thermo Fisher Scientific, clone OTI4E7; 1:3,000 dilution) and anti-actin antibody (Merck, MAB1501R, clone C4; 1:1,000 dilution) and secondary antibodies including anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074; 1:4,000 dilution) and goat anti-mouse HRP-conjugated antibody (Bethyl, A90-116P; 1:2,000 dilution) were diluted in 5% skim milk in TBS-T before use.

Techniques: Transfection, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing, Transduction, shRNA, Knockdown, Fluorescence

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: a , Schematics of SH2 domains of STAT5A in dimeric complexes. LOF and GOF substitutions are indicated in magenta and green, respectively. Structures were predicted using AlphaFold simulation. b , Representative fast FLIM images of HEK-Blue IL-2 cells expressing LOF and GOF STATeLight5A biosensors before and after IL-2 stimulation. c , Quantification of mNG fluorescence lifetime in cells expressing LOF and GOF STATeLight5A. Box plots show the median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. From left to right, n = 50, 33, 32, 30, 20 and 14 cells. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: **** P = 8.22 × 10 −17 (WT), P = 0.368 (NS; Y694F), P = 0.694 (NS; W641A), P = 0.368 (NS; F706A), **** P = 4.35 × 10 −11 (S710F) and **** P = 1.99 × 10 −7 (Y665F). d , Western blot analysis of HEK-Blue IL-2 cells expressing LOF and GOF STATeLight5A biosensors using anti-STAT5A and anti-pSTAT5 (pY694) antibodies. Dimeric pSTAT5 observed for LOF mutants stemmed from endogenous STAT5B of HEK-Blue IL-2 cells. e , Quantification of mNG fluorescence lifetime in cells coexpressing GOF mutants and PTPN1. Box plots show the median (center line), 25th and 75th percentiles (box limits) and whiskers extending 1.5× the interquartile range. From left to right, n = 35, 32 and 20 cells. A two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted P values: * P = 0.028, **** P = 1.64 × 10 −12 (S710F) and **** P = 3.48 × 10 −10 (Y665F). f , Western blot analysis using anti-pSTAT5 antibodies in indicated GOF mutants.

Article Snippet: The membrane was blocked with 5% skim milk in 0.1% Tween-20 in Tris-buffered saline pH 7.4 (TBS-T) for 2 h, followed by incubation with primary antibodies overnight and subsequently secondary anti-horseradish peroxidase (HRP) antibodies for 2 h. Primary antibodies including anti-pSTAT5 (Y694) antibody (Cell Signaling, 9351; 1:3,000 dilution), anti-STAT5A monoclonal antibody (TrueMAB, Thermo Fisher Scientific, clone OTI4E7; 1:3,000 dilution) and anti-actin antibody (Merck, MAB1501R, clone C4; 1:1,000 dilution) and secondary antibodies including anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074; 1:4,000 dilution) and goat anti-mouse HRP-conjugated antibody (Bethyl, A90-116P; 1:2,000 dilution) were diluted in 5% skim milk in TBS-T before use.

Techniques: Expressing, Fluorescence, Western Blot

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( a ) Schematic representation of cancer-associated STAT5B mutations. Blue, SH2 domain; violet, linker domain (LD); orange, DNA-binding domain (DBD); and green, coiled coil domain (CCD). ( b ) Flow cytometry plots showing pSTAT5 in cancer-associated STAT5B mutants, expressed in HEK-Blue TM IL-2 cells. ( c ) Quantification of pSTAT5 in cells expressing cancer-associated STAT5B gain-of-function mutations. ( d ) Western blot analysis of HEK-Blue TM IL-2 cells expressing full-length cancer-associated STAT5B mutants. ( e ) Quantification of mNG fluorescence lifetime in cells expressing STATeLight5B biosensors harboring cancer-associated mutations. From left to right, n = 19, 18, 31, 27, 33, 27, 23, 30, and 28 cells. Boxplots show median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 1.56 × 10 −5 (Y712E), **** p = 5.50 × 10 −6 (Y665F), **** p = 2.11 × 10 −6 (N642H), **** p = 4.22 × 10 −7 (A510V), **** p = 1.81 × 10 −11 (E438Q), **** p = 4.93 × 10 −10 (S434L), **** p = 3.20 × 10 −9 (N418K), **** p = 1.85 × 10 −13 (P267A), **** p = 4.13 × 10 −12 (WT). ( f ) HEK-Blue TM IL-2 reporter assay in cells expressing cancer-associated STAT5B mutants and using titrated concentrations of IL-2. Shown are mean ± SEM of 3 biological replicates. ( g ) Quantification of mNG fluorescence lifetime in cells co-expressing STATeLight5B biosensors with cancer-associated mutations and protein tyrosine phosphatase non-receptor type 1 (PTPN1). From left to right, n = 17, 16, 47, 27, 19, 20, 19, 25, and 52 cells. Boxplots show median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 4.01 ×10 −6 (Y712E), **** p = 1.20 × 10 −7 (Y665F), **** p = 1.25 × 10 −14 (N642H), **** p = 7.70 × 10 −5 (A510V), *** p = 7.92 × 10 −4 (E438Q), ** p = 4.80 × 10 −3 (S434L), **** p = 1.64 × 10 −5 (N418K), 0.803, ns (P267A), **** p = 1.70 × 10 −8 (WT). ( h ) Western blot analysis of HEK-Blue TM IL-2 cells co-expressing full-length cancer-associated STAT5B mutants and PTPN1.

Article Snippet: The membrane was blocked with 5% skim milk in 0.1% Tween-20 in Tris-buffered saline pH 7.4 (TBS-T) for 2 h, followed by incubation with primary antibodies overnight and subsequently secondary anti-horseradish peroxidase (HRP) antibodies for 2 h. Primary antibodies including anti-pSTAT5 (Y694) antibody (Cell Signaling, 9351; 1:3,000 dilution), anti-STAT5A monoclonal antibody (TrueMAB, Thermo Fisher Scientific, clone OTI4E7; 1:3,000 dilution) and anti-actin antibody (Merck, MAB1501R, clone C4; 1:1,000 dilution) and secondary antibodies including anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074; 1:4,000 dilution) and goat anti-mouse HRP-conjugated antibody (Bethyl, A90-116P; 1:2,000 dilution) were diluted in 5% skim milk in TBS-T before use.

Techniques: Binding Assay, Flow Cytometry, Expressing, Western Blot, Fluorescence, Reporter Assay

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: ( a ) Quantification of mNG fluorescence lifetime in cells expressing C-terminally labelled full-length STAT5A biosensor variants, including WT (black, n = 27 cells), loss-of-function (LOF) mutant (Y694F, magenta, n = 24 cells), and gain-of-function (GOF) mutant (S710F, green, n = 35 cells). Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: **** p = 6.17 × 10 −15 (WT), 0.282, ns (Y694F), **** p = 7.89 × 10 −16 (S710F). ( b ) Western blot analysis using anti-STAT5A and anti-pSTAT5 antibodies in HEK-Blue TM IL-2 cells expressing the full-length STAT5A biosensor variants shown in ( a ). ( c ) Western blot analysis using anti-STAT5A and anti-pSTAT5 antibodies in HEK-Blue™ IL-2 cells co-expressing PTPN1 and full-length STAT5A biosensor variants, including WT (black) and GOF S710F mutant (green). ( d ) mNG fluorescence lifetime measured in cells co-expressing PTPN1 and full-length STAT5A biosensor variants, including WT (black, n = 23 cells) and the GOF S710F mutant (green, n = 25 cells). Boxplots show the median (center line), 25 th and 75 th percentiles (box limits), and whiskers extending 1.5 × the interquartile range. Two-sided Wilcoxon’s rank-sum test was used to calculate FDR-adjusted p values: 0.965, ns (WT), 0.242, ns (S710F).

Article Snippet: The membrane was blocked with 5% skim milk in 0.1% Tween-20 in Tris-buffered saline pH 7.4 (TBS-T) for 2 h, followed by incubation with primary antibodies overnight and subsequently secondary anti-horseradish peroxidase (HRP) antibodies for 2 h. Primary antibodies including anti-pSTAT5 (Y694) antibody (Cell Signaling, 9351; 1:3,000 dilution), anti-STAT5A monoclonal antibody (TrueMAB, Thermo Fisher Scientific, clone OTI4E7; 1:3,000 dilution) and anti-actin antibody (Merck, MAB1501R, clone C4; 1:1,000 dilution) and secondary antibodies including anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074; 1:4,000 dilution) and goat anti-mouse HRP-conjugated antibody (Bethyl, A90-116P; 1:2,000 dilution) were diluted in 5% skim milk in TBS-T before use.

Techniques: Fluorescence, Expressing, Mutagenesis, Western Blot

Journal: Nature Chemical Biology

Article Title: Real-time visualization of STAT activation in live cells using genetically encoded biosensors

doi: 10.1038/s41589-025-02012-0

Figure Lengend Snippet: Human PBMCs or HEK-Blue TM IL-2 cells were pre-treated with titrated amounts of Filgotinib or Upadacitinib, followed by stimulation with IL-2. ( a ) Gating strategy to identify CD4 + T cells in human PBMCs. ( b ) Representative pSTAT5 histograms of CD4 + T cells treated with Filgotinib or Upadacitinib. ( c ) Percentage of pSTAT5 + cells in CD4 + T cells treated with Filgotinib (yellow) or Upadacitinib (purple). Data are represented as mean ± SEM of n = 3 replicates. ( d ) Dose-response of HEK-Blue TM IL-2 cells treated with Filgotinib (yellow) or Upadacitinib (purple) using HEK-Blue TM IL-2 reporter assay. Data are represented as mean ± SEM (n = 3 replicates). ( e) Time-course of mNG fluorescence lifetime in HEK-Blue™ IL-2 cells expressing STATeLight5A, untreated (n = 75 cells) or treated with Upadacitinib (n = 39 cells) or Filgotinib (n = 33 cells) and stimulated with IL-2. Data are shown as mean ± SEM.

Article Snippet: The membrane was blocked with 5% skim milk in 0.1% Tween-20 in Tris-buffered saline pH 7.4 (TBS-T) for 2 h, followed by incubation with primary antibodies overnight and subsequently secondary anti-horseradish peroxidase (HRP) antibodies for 2 h. Primary antibodies including anti-pSTAT5 (Y694) antibody (Cell Signaling, 9351; 1:3,000 dilution), anti-STAT5A monoclonal antibody (TrueMAB, Thermo Fisher Scientific, clone OTI4E7; 1:3,000 dilution) and anti-actin antibody (Merck, MAB1501R, clone C4; 1:1,000 dilution) and secondary antibodies including anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074; 1:4,000 dilution) and goat anti-mouse HRP-conjugated antibody (Bethyl, A90-116P; 1:2,000 dilution) were diluted in 5% skim milk in TBS-T before use.

Techniques: Reporter Assay, Fluorescence, Expressing