Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Following stimulation, the cell lysates were analyzed by Western blotting using an anti-pSTAT5 antibody (Cell Signaling).

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

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: Biomedical Reports

Article Title: Hemp seed extract as a catalyst for cell proliferation via IGF-1 and growth hormone signaling: Increased natural regenerative potential of muscles

doi: 10.3892/br.2025.2082

Figure Lengend Snippet: HS extracts enhances expression of nuclear pSTAT5b and its binding to the IGF-1 promoter. (A) Western blotting showing the protein expression of JAK2, pJAK2, STAT5b and pSTAT5 following the treatment of C2C12 and C3H10T1/2 cells with 100 and 200 µg/ml HS extract for 24 h. (B) Western blotting showing the protein expression of GHR, IGF-1, pIGF-1Rβ and IGF-1Rβ following the treatment of C2C12 and C3H10T1/2 cells with 100 and 200 µg/ml HS extracts for 24 h. (C) Relative STAT5b/IGF-1 complex binding in C2C12 and C3H10T1/2 cells treated with 100 and 200 µg/ml HS extracts for 24 h by chromatin immunoprecipitation assay. # P<0.001 vs. control. HS, hemp seed; p, phosphorylated; IGF-1R, insulin-like growth factor-1receptor; GHR, growth hormone receptor.

Article Snippet: Primary antibodies against pSTAT5 (cat. no. #9351), JAK2 (cat. no. #3230), pJAK2 (cat. no. #3776), pIGF-1Rβ (cat. no. #3012s) and cleaved caspase3 (cat. no. #9661s) were obtained from Cell Signaling Technology, Inc. IGF-1 antibody (cat. no. ab9572) was purchased from Abcam.

Techniques: Expressing, Binding Assay, Western Blot, Chromatin Immunoprecipitation, Control

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