Fig 2: Comparison of in silico (left) with in vivo (right) dynamics of EGFR degradation pathway. A, B. The temporal dynamics of EGFR-bound Cbl-b and ubiquitinated EGFR. Extracted proteins were normalized to the initial expression amount of EGFR, and then subjected to EGFR immunoprecipitation. Immunoblottings were performed to detect co-immunoprecipitated Cbl-b and ubiquitinated EGFR. Error bars represent the deviations of two independent experiments. The simulated results were normalized to the initial abundance of EGFR predicted in silico. C. The temporal dynamics regarding the total amount of EGFR. EGF-stimulated cell lysates were applied to immunoblots to detect total EGFR protein. Band intensities and simulated results were normalized to the value at zero time point for each cell type. Error bars represent standard deviations of three independent experiments. The raw data on the immunoblot experiments can be found in Figure S4.

Fig 3: siRNA-mediated silencing of human CBLB is effective and leads to hyper-responsiveness of CD8+ T cells.Human CD8+ T cells were nucleofected with CBLB siRNA #10 or control siRNA and stimulated with anti-CD3 and anti-CD28 as indicated. Similar results were obtained with non-overlapping siRNA oligonucleotide #11 (not shown). (A) Supernatants were taken two days post-transfection and the amount of IFN? secreted was measured by BioPlex technology. Means ± SEM of two independent experiments are shown. p = 0.01 (CD8+ CBLB siRNA versus CD8+ control siRNA, double stimulated). (B) Human CD8+ T cells were nucleofected with CBLB siRNA or control siRNA and stimulated with anti-CD3 and anti-CD28 in the presence or absence of TGFß. Supernatants were taken two days post-transfection and the amount of IFN? secreted was measured by BioPlex technology. IFN? levels of each group without TGFß were set 100%. Representative results of two independent experiments are shown. Silencing efficacy of cblb siRNA was analyzed by (A) immunoblotting (One experiment representative of three experiments is shown) or (C) qRT-PCR. Means ± SEM of two independent experiments are shown.

Fig 4: CBL depletion or inactivation increases JAK2 level and promotes cell growth. (A) TF-1/hMPL cells with single or double depletion of CBL and CBL-b were analyzed for JAK2 levels by Western blot. (Luc) Luciferase; (gRNA) guide RNA. (B–D) Cells were cultured in different concentrations of TPO (B), GM-CSF (C), or ruxolitinib in the presence of TPO (D). Cell numbers after 3 d of culture were determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) absorbance. (D) The left panel shows the absolute MTT values, while the right panel shows the relative MTT values normalized to 0 nM ruxolitinib in each cell line. (E) TF-1/MPL cells stably expressing either control vector, CBL wild type, or the E3-dead mutant C381A were examined for JAK2 levels by Western blot. (exp) Exposure. (F–H) MTT assay of the indicated TF-1 cell lines cultured in different concentrations of TPO (F), GM-CSF (G), or ruxolitinib in the presence of TPO (H). (H) The left panel shows the absolute MTT values, while the right panel shows the relative MTT values normalized to 0 nM ruxolitinib in each cell line. Error bars denote mean ± SD. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Fig 5: miR-1323 can promotes the migration of A549 cells through inhibiting Cbl-b. (A) A schematic view of miR-1323 predicted target genes through four miRNA databases of different mechanisms. The A549 cells were transfected with miR-1323 mimic or NC, (B) WB detected the expression of proteins and (C) Elisa detected the secretory level of IL-6 in A549 cells. (D) The bindng site of miR-1323 to Cbl-b 3'UTR. (E) The A549 cells were co-transfected with pMirTarget-Cbl-b WT (or MUT) plasmid, miR-1323 mimic (or NC), and pRL-TK. The dual luciferase reporter assay detected the activity of luciferase. The histogram shows the relative activity of luciferase. t-test was used to assess statistically signifificant differences between groups.Mean ± SD, results of three independent experiments, *p < 0.05.