Figure 1: c-Src controls IL-6 expression by modulation of STAT3 activation. | Nature Communications

Figure 1: c-Src controls IL-6 expression by modulation of STAT3 activation.

From: c-Src and IL-6 inhibit osteoblast differentiation and integrate IGFBP5 signalling

Figure 1

(a) Mouse calvarial osteoblasts were treated with DMSO or 2 μM PP1 in a time-course experiment. RT–PCR and relative quantification of Il-6 expression, normalized with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) expression (mean±s.d., n=3, **P<0.01, Student's t-test). (b) ELISA assay of IL-6 protein accumulation in conditioned media in the time-course experiment shown in a (mean±s.d., n=3, *P<0.05, **P≤0.005, Student's t-test). (c) Mouse calvarial osteoblasts were treated with 100 nM c-Src–siRNA. RT–PCR of Il-6 expression, normalized versus Gapdh expression (mean±s.d., n=3, **P<0.005, Student's t-test), and western blot (WB) analysis to control total c-Src protein downregulation, normalized versus actin. (d) Mouse calvarial osteoblasts were electroporated with an empty vector, a WT and a dominant negative (DN) variant of c-Src. RT–PCR of Il-6 expression, normalized versus Gapdh expression (mean±s.d., n=3, *P<0.05, Student's t-test), and WB analysis to control total c-Src protein downregulation and total phospho-tyrosine status, normalized versus actin. (e) STAT proteins, both total and phosphorylated in activating tyrosine residues, were evaluated in DMSO- or PP1-treated osteoblasts, normalized versus actin. (f) Immunofluorescence analysis for total STAT3 in primary mouse osteoblasts treated for 24 h with 2 μM PP1. Results are representative of three independent experiments. Scale bar, 50 μm. (g) Immunofluorescence analysis for phospho-Y705-STAT3 protein in primary mouse osteoblasts treated for 24 h with 2 μM PP1. Results are representative of three independent experiments. Scale bar, 50 μm. (h) Immunoprecipitation (IP) of osteoblast lysates with total c-Src antibody followed by a WB with a total STAT3 antibody. Results are representative of three independent experiments. TCL, total cell lysate. (i) Stripping of the membrane shown in h and staining with a P-Y705-STAT3 antibody. (j) Mouse calvarial osteoblasts were treated with 100 nM STAT3–siRNA. RT–PCR of Il-6 expression, normalized versus Gapdh (mean±s.d., n=3, **P<0.005, Student's t-test), and WB analysis to control total c-Src protein downregulation, normalized versus actin. CTL, control siRNA; DAPI, 4,6-diamidino-2-phenylindole; IgG, immunoglobulin-G.

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