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. 2005 Mar 21;102(13):4700–4705. doi: 10.1073/pnas.0409894102

Fig. 5.

Fig. 5.

STA-21 inhibits Stat3 translocation and dimerization in breast carcinoma cells. The MDA-MB-435s cells cotransfected with pCMV-Stat3-Flag and pCMV-Stat3-HA plasmid were exposed to 20 μM STA-21 compound for 24 h, then fixed with 100% methanol. After the fixed cells were stained with anti-HA (rabbit, Santa Cruz Biotechnology) and/or anti-Flag (mouse, Sigma) Abs, secondary anti-rabbit IgG-FITC and/or anti-mouse IgG-rhodamine Abs were added. The cells were observed by using a fluorescence microscope. (A) Untransfected and untreated MDA-MB-435s cells. (B and E) Untreated (B) and STA-21-treated (E) cells cotransfected by pCMV-Stat3-Flag and pCMV-Stat3-HA plasmids were immunostained with anti-Flag IgG-rhodamine. (C and F) Untreated (C) and STA-21 treated (F) transfected cells were immunostained with anti-HA IgG-FITC. (D and G) The transfected cells were coimmunostained with both anti-HA IgG-FITC and anti-Flag IgG-rhodamine. (D) Untreated cells showed bright orange color. (G) STA-21 treated cells showed weak orange staining and separate green and red color. (Magnification: ×400.) (H) The MDA-MB-435s cells were cotransfected with pCMV-Stat3-Flag and pCMV-Stat3-HA plasmids and were exposed to 20 μM STA-21 compound for 24 h, and then cell lysates were immunoprecipitated with anti-HA or anti-Flag Abs, respectively, as described previously. Cells were resolved on 10% SDS/PAGE and then immunoblotted with anti-HA, anti-Flag or anti-Sat3 Abs as described in Materials and Methods.