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Figure 4 | Molecular Cancer

Figure 4

From: The haematopoietic GTPase RhoH modulates IL3 signalling through regulation of STAT activity and IL3 receptor expression

Figure 4

Low RhoH expression levels enhance STAT5 activity. (A) Control cells, siRhoH and RhoH expressing cells were starved for 3 h in the absence of cytokine and FBS before stimulation with 50 ng/ml IL3. Cells were lysed and STAT5 was immunoprecipitated. Tyrosine phosphorylated STAT5 and total STAT5 were detected by enhanced chemiluminescence using specific antibodies. Quantification of pSTAT5 levels was done after normalisation to total STAT5 levels in control cells and is presented as induction of phosphorylation compared to the corresponding unstimulated sample. Statistical significance was analysed using the 's t-test (mean ± SD, n = 2; *P ≤ 0.05). (B) To verify equal expression levels of STAT5 protein in all cell lines, STAT5 was detected from whole cell lysates and quantified after normalisation to β-actin levels of control cells (mean ± SD, n = 3). (C) pSTAT5 levels were measured by intracellular FACS staining at 0 and 10 min of stimulation of previously starved cells with 50 ng/ml IL3. Representative graphs from one out of three independently performed experiments are shown. (D) CD123 surface expression of control cells, siRhoH and RhoH cells determined by FACS analysis using a CD123-PE antibody. The left panel shows a typical example and the right panel summarises the data of the mean fluorescence signal and the statistical analysis (mean ± SD, n = 3, *P ≤ 0.05). (E) Quantification of IRF-1 mRNA expression levels of control cells and siRhoH-transduced cells was done by quantitative real-time PCR using GAPDH as a reference gene. Data were normalised to the expression in control cells and are presented as fold induction of the IRF-1 gene compared to control cells (mean ± SD, n = 3; *P ≤ 0.05).

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