Journal: Cell Communication and Signaling : CCS

Article Title: E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

doi: 10.1186/s12964-024-01636-4

Figure Lengend Snippet: RNF128 negatively regulates IL-3-triggered signaling. ( A , B ) Effects of Rnf128 on GM-CSF-induced phosphorylation of Stat5 and transcription of downstream genes. Wild-type and Rnf128 -deficient BMDMs were treated with GM-CSF (10 ng/ml) or not for the indicated times before immunoblotting analysis and qPCR analysis. ( C , D ) Effects of Rnf128-deficient on Il-3-induced phosphorylation of Stat5. Wild-type and Rnf128 -deficient BMDMs were either untreated or treated with Il-3 (25 ng/mL) for the specified durations. Subsequently, immunoblotting analysis was conducted to detect p-Stat5 levels, with quantification illustrating the ratio of p-Stat5 to Stat5 ( D ). ( E ) Effects of Rnf128-deficient on Il-3-induced Id1 , Pim1 and Cd69 genes. ( F , G ) Effects of RNF128 knockdown on IL-3-induced phosphorylation of Stat5. RNF128 knockdown and control TF-1 cells were subjected to overnight starvation, followed by stimulation with IL-3 (20 ng/mL) for the indicated durations. Subsequently, immunoblotting analysis was conducted for p-STAT5, and the quantification of p-STAT5/STAT5 ratios is presented ( G ). ( H ) Effects of RNF128 knockdown on the transcription of the ID1, PIM1 AND CD69 genes induced by IL-3. * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical analysis was performed using two-way ANOVA with Dunnett’s post-hoc test

Article Snippet: Human TF-1 cells, generously provided by Procell Life Science and Technology, were cultured in RPMI-1640 (Corning) medium supplemented with 2 ng/mL GM-CSF and 10% FBS.

Techniques: Western Blot