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. 2009 May 22;37(13):4393–4406. doi: 10.1093/nar/gkp398

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© 2009 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — RFXB silencing favors the TGF-β antagonism of IFN-γ induced collagen repression. (A) HEK293 cells were transfected with FLAG-tagged RFXB or RFXBSV plasmids in the presence of shB, shB/SV or an empty vector (EV). Silencing efficiency was measured by Western blot. (B, C, D) HASMCs were infected with viral stocks carrying shB, shB/SV or an EV followed by treatment with IFN-γ and/or TGF-β as indicated. Message (B) and protein (C) levels of collagen type I were assessed by real-time PCR and Western blot, respectively as described in Figure 1. ChIP assays (D) with anti-AcH3, -HDAC2 and –RFX5 antibodies were performed as described in Figure 5. (E) A collagen type I promoter construct (pH 20) was co-transfected with expression constructs for HDAC2 into HASMCs along with shB, shB/SV or an EV as indicated, followed by treatment with IFN-γ and/or TGF-β. Luciferase activities were normalized by both protein concentration and GFP fluorescence, and presented as average ± SD (*P < 0.05).

Figure 6. — RFXB silencing favors the TGF-β antagonism of IFN-γ induced collagen repression. (A) HEK293 cells were transfected with FLAG-tagged RFXB or RFXBSV plasmids in the presence of shB, shB/SV or an empty vector (EV). Silencing efficiency was measured by Western blot. (B, C, D) HASMCs were infected with viral stocks carrying shB, shB/SV or an EV followed by treatment with IFN-γ and/or TGF-β as indicated. Message (B) and protein (C) levels of collagen type I were assessed by real-time PCR and Western blot, respectively as described in Figure 1. ChIP assays (D) with anti-AcH3, -HDAC2 and –RFX5 antibodies were performed as described in Figure 5. (E) A collagen type I promoter construct (pH 20) was co-transfected with expression constructs for HDAC2 into HASMCs along with shB, shB/SV or an EV as indicated, followed by treatment with IFN-γ and/or TGF-β. Luciferase activities were normalized by both protein concentration and GFP fluorescence, and presented as average ± SD (*P < 0.05).