anti cd127  (Miltenyi Biotec)

Journal: iScience

Article Title: Translation factor eIF4G2 directs CD8 + T cell lineage commitment by selectively enabling the IL-7 receptor response

doi: 10.1016/j.isci.2026.115313

Figure Lengend Snippet: eIF4G2 specifically sustains surface expression of the IL-7 receptor (A–C) Expression of IL-7Rα (CD127). (A) Representative histogram and (B) quantification of CD127 median fluorescence intensity (MFI) on CD4 + CD8 lo transitional cells ( n = 3, ∗∗ p < 0.01). (C) MFI of CD127 on DP, CD4 SP, and CD8 SP thymocytes ( n = 3, ns p > 0.05, ∗∗∗∗ p < 0.0001). (D–F) Expression of the common γc (CD132). (D) Representative histogram and (E) quantification of CD132 MFI on CD4 + CD8 lo transitional cells ( n = 4, ∗ p < 0.05). ppp(F) MFI of CD132 on DP, CD4 SP, and CD8 SP thymocytes ( n = 3, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (G and H) Expression of IL-4Rα (CD124) expression detection on CD4 + CD8 lo transitional cells. (G) Representative histogram and (H) quantification of CD124 MFI on CD4 + CD8 lo transitional cells ( n = 3, ns p > 0.05). (I and J) Expression of GP130 on CD4 + CD8 lo transitional cells. (I) Representative histogram and (J) quantification of GP130 MFI on CD4 + CD8 lo transitional cells ( n = 3, ∗∗ p < 0.01). (K) Quantification of CD124 MFI on DP, CD4 SP and CD8 SP ( n = 3, ns p > 0.05). (L–N) Expression of IL-2Rα (CD25). (L) Representative histogram and (M) quantification of CD25 MFI on CD4 + CD8 lo transitional cells ( n = 3, ns p > 0.05). (N) MFI of CD25 on DP, CD4 SP, and CD8 SP subsets ( n = 3, ns p > 0.05). Data are representative of at least two independent experiments. Bar graphs show mean ± SEM and unpaired Students’ t test was used to perform the statistical analysis.

Article Snippet: Membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in blocking buffer: eIF4G2 (CST, 3468S), β-actin (CST, 3700S), eIF4G1 (Proteintech, 15704-1-AP), IL-7Rα (Proteintech, 17626-1-AP), γc (Proteintech, 11409-1-AP), STAT5 (Proteintech, 13179-1-AP), phospho STAT5 (CST, 4322T), STAT6 (Proteintech, 51073-1-AP), mouse phospho STAT6 (CST, 56554S).

Techniques: Expressing, Fluorescence

Journal: iScience

Article Title: Translation factor eIF4G2 directs CD8 + T cell lineage commitment by selectively enabling the IL-7 receptor response

doi: 10.1016/j.isci.2026.115313

Figure Lengend Snippet: eIF4G2 post-transcriptionally sustains γc expression via its mRNA UTRs (A–C) Analysis in primary CD4 + CD8 lo transitional thymocytes. (A) Western blot analysis of γc and IL-7Rα protein levels. (B and C) Quantitative RT-PCR analysis of Il2rg (B) and Il7r (C) mRNA levels ( n = 3, ns p > 0.05, ∗ p < 0.05). (D–H) Mechanistic dissection in 293T cells. (D) Western blot of γc protein in control and EIF4G2 knockdown 293T cells transfected with an IL2RG coding sequence construct containing its native 5′ and 3′ UTRs. (E) Corresponding IL2RG mRNA levels measured by RT-qPCR ( n = 3, ns p > 0.05) . (F and G) Assessment of γc protein stability ( n = 3, ns p > 0.05). (F) Representative western blots and (G) quantification of γc protein levels over time following cycloheximide (CHX) treatment in si-control and si- EIF4G2 293T cells ( n = 3, ns p > 0.05). (H) Western blot of γc protein in si-control and si- EIF4G2 293T cells transfected with an IL2RG CDS construct lacking UTRs. Data are representative of at least two independent experiments. Bar graphs show mean ± SEM and unpaired Students’ t test was used to perform the statistical analysis.

Article Snippet: Membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in blocking buffer: eIF4G2 (CST, 3468S), β-actin (CST, 3700S), eIF4G1 (Proteintech, 15704-1-AP), IL-7Rα (Proteintech, 17626-1-AP), γc (Proteintech, 11409-1-AP), STAT5 (Proteintech, 13179-1-AP), phospho STAT5 (CST, 4322T), STAT6 (Proteintech, 51073-1-AP), mouse phospho STAT6 (CST, 56554S).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Dissection, Control, Knockdown, Transfection, Sequencing, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in cell lines. ( A ) mRNA normalized TPM of IL4Rα from Human Protein Atlas for Daudi and SiHa cell lines. ( B ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL4-Bevacizumab in Daudi and SiHa cell lines. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. Means ± SEM of three biological replicates are shown. ( C ) mRNA normalized TPM of IL7R from Human protein atlas for Daudi and SiHa cell lines. ( D ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL7-Bevacizumab in Daudi and SiHa cell lines. Curves are best fits for three-parameter nonlinear regressions. Mean values ± SEM are from three biological replicates. ( E ) Diagram of coculture experiments. Daudi cells (gray) and SiHa cells (purple) are coincubated with VEGF-647 and either IL4-Bevacizumab kineTAC (green) or IL7-Bevacizumab kineTAC (blue). Cell type–specific receptor expression allows for cell-specific internalization of VEGF using the respective kineTAC. ( F ) Representative flow cytometry data from coculture experiment. Daudi and SiHa cells in equal amounts were incubated for 24 h with 25 nM VEGF-647, and 0.3 nM IL4-Bevacizumab or 10 nM IL7-Bevacizumab. Gates show thresholds for VEGF positivity, defined as approximately 1% of total cells for VEGF-647 only. Top row: FITC + Daudi GFP . Bottom row: FITC − SiHa. Percentages in gate are displayed. ( G ) Fold change in VEGF-647 median fluorescence intensity in the coculture Daudi and SiHa experiment from F . Mean fold change over 25 nM VEGF-647 alone ± SEM is presented. The asterisk represents a discovery (q < 1%) using the false discovery rate to correct for multiple comparisons. ( H ) Coculture VEGF-647 percent positivity (same gate as in F ) as a dose response of IL4-Bevacizumab. Mean values and ± SEM are from three biological replicates. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. ( I ) same as in H , but for IL7-Bevacizumab.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in primary lymphocytes and PBMCs. ( A ) Diagram depicting tissue-specific expression of potential kineTAC receptors. ( B ) tSNE analysis of IL4Rα expression from scRNA seq of two PBMC donors, obtained from the Immune Cell Atlas ( 57 ). Notice the prominent naïve B cell cluster of high IL4Rα expressors (green). ( C ) tSNE analysis of IL7R expression from the same dataset. Notice primarily T cell restricted expression of IL7R (blue). ( D ) Immune Cell Atlas cluster annotations for primary cell lineages, colored by lineage as in legend to the right. ( E ) Raw VEGF-647 median fluorescence intensities across two donors for isolated CD19+ cells from four technical replicates. Cells were incubated with 25 nM VEGF-647 and 10 nM of indicated kineTAC or isotype. ( F ) same as E , but using CD3+ T-cells. ( G and H ) VEGF-647 with PBMC components. 10 nM of the indicated kineTAC or isotype was added to 25 nM VEGF-647 along with 200 k cells/well PBMCs and allowed to internalize for 24 h. Percent VEGF positive was defined by the no VEGF-647 condition ( SI Appendix , Fig. S6 ). Analyzed by repeated measures donor-paired one-way ANOVA, with P values for Dunnett’s correction for multiple comparisons to the kineTAC of interest (IL4-Beva for B cells, IL7-Beva for T cells) (* P < 0.05, ** P < 0.01). ( G ) Analysis of B cell gate and h analysis of T cell gate.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Fluorescence, Isolation, Incubation