Journal: bioRxiv

Article Title: AXL mediates mast cell survival and resistance to tyrosine kinase inhibitors in mastocytosis

doi: 10.1101/2025.11.03.686205

Figure Lengend Snippet: (A) Proliferation kinetics of ROSA KIT WT, ROSA KIT D816V, and (B) BMMC KIT D814V transduced with lentiviral vectors AXL WT (Blue), AXL L197M (Pink) and Ctrl cells (green), monitored by incucyte live-cell imaging. Confluence (%) is plotted against time (in hours). (C) Immunoblot of AXL in ROSA KIT WT and ROSA KIT D816V cells transduced with AXL WT -tdTomato , AXL L197M - tdTomato lentiviral vectors, or control cells. (D) Colony formation was assayed in ROSA KIT D816V cells transduced with the same lentiviral vectors and control cells. (E) Immunoblots of AXL, total and phosphorylated STAT5, STAT3, FAK and p38α in transduced ROSA KIT- D816V. β-actin or HSP70 were used for normalization. Statistical analyses were performed by one-way ANOVA: * p<0.05; **p<0.01; ***p<0.001. Data are representative of 3 independent experiments. é

Article Snippet: ROSA KIT D816V cells expressing AXL WT-tdTomato, AXL L197M-tdTomato, or control were analyzed for the expression of STAT3 (9139, Cell Signaling), pSTAT3 (Y705) (9145, Cell Signaling), STAT5 (25656, Cell Signaling), pSTAT5 (Y694) (9359, Cell Signaling), FAK (3285, Cell Signaling), pFAK (Y397) (8556, Cell Signaling), p38α (sc-81621, Santa cruz), p-p38 (sc-7973, Santa cruz).

Techniques: Transduction, Live Cell Imaging, Western Blot, Control

Journal: The Application of Clinical Genetics

Article Title: Hemizygous IL2RG Variants Impair IL-2-Induced STAT5 Phosphorylation and Transcriptional Activity Causing X-Linked Severe Combined Immunodeficiency

doi: 10.2147/TACG.S525027

Figure Lengend Snippet: Reduced IL2RG expression on the cell surface affects IL-2-mediated signal transduction. ( A ) IL2RG mutant protein expression in HEK293 cells, as determined by Western blotting. ( B ) Co-IP analysis showed significantly reduced binding between JAK3 and the three IL2RG mutant proteins (p.R190P, p.L172P, and p.T364I), while the p.T73P mutant protein exhibited partial interaction with JAK3. ( C ) Flow cytometry analysis demonstrated that the surface expression of mutant IL2RG proteins was significantly reduced in HEK293 cells compared to that of wild-type IL2RG. ( D ) After stimulation with 1000 U/mL IL-2, STAT5 phosphorylation was substantially decreased in cells expressing mutant IL2RG proteins compared to wild-type IL2RG. ( E ) Luciferase reporter assays showed a significant reduction in STAT5 transcriptional activity in cells expressing the mutant IL2RG proteins compared to those expressing wild-type IL2RG. Cells in the blank control group were untransfected with any plasmid. Data are representative of three or four independent experiments, results are represented as mean ± SD, and statistical analyses were carried out via t test. (** p <0.01; *** p <0.001; **** p <0.0001).

Article Snippet: Membranes were incubated overnight at 4 °C with primary antibodies against Flag-tag, Myc-Tag (1:1000, Abways Technology, AB0001, Shanghai, China), Stat5 (D2O6Y) Rabbit mAb (1:1000, Cell Signaling Technology, 94205, Danvers, MA, USA), Phospho-Stat5 (Tyr694) (D47E7) XP Rabbit mAb (1:1000, Cell Signaling Technology, 4322, Danvers, MA, USA), and GAPDH (1:1000, Affinity Biosciences, T0004, Liyang, Jiangsu, China).

Techniques: Expressing, Transduction, Mutagenesis, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Flow Cytometry, Phospho-proteomics, Luciferase, Activity Assay, Control, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: JAK3 Y841 Autophosphorylation Is Critical for STAT5B Activation, Kinase Domain Stability and Dimer Formation.

doi: 10.3390/ijms241511928

Figure Lengend Snippet: Figure 2. JAK3 Y841 and the homologous JAK1 Y894 are critical for the activation of STAT5B. (a) Hek293 cells were transfected with 10 µg WT (lane a) or mutant JAK3 plasmids (lanes b–i) and incubated for 48 h at 37 ◦C. Cells were harvested after 48 h, immunoprecipitated for JAK3 and Western blotted for total pY. Shown is a representative blot from n = 2 independent experiments. Individual points for replicate experiments are provided in Figure S2a. (b) Hek293 cells were transfected with 2 µg of WT (lane a) or mutant JAK3 plasmids (lanes b–i) with 2 µg of STAT5B plasmids (lanes b–i) and harvested after 48 h at 37 ◦C. Western blot was performed using anti-pYSTAT5 and reblotted for total STAT5 to confirm the equal protein expression and loading of STAT5B. Shown is a representative blot from n = 3 independent experiments. (c) Densitometry analysis using LI-COR Image Studio Digits

Article Snippet: For the JAK3/STAT5 activation studies, HEK293 cells were transfected with 2 μg of pcDNA3.1/human JAK3 plasmid [19] alone or with 2 μg of STAT5B plasmid (OriGene, Rockville, MD, USA) per confluent in 10 cm dishes.

Techniques: Activation Assay, Transfection, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Expressing