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. 2012 Sep 4;122(10):3742–3754. doi: 10.1172/JCI62727

Figure 6. HSF1 is overexpressed and activated in human MPNST cell lines lacking neurofibromin.

Figure 6

(A) Immunoblotting of cell lines with complete loss of neurofibromin showed elevated p–C-RAF, p-MEK1/2, total HSF1, p-Ser362, and HSP72 levels compared with lines that maintain full-length neurofibromin expression. (B) IF staining confirmed HSF1 overexpression in cells without neurofibromin. Insets show nuclear HSF1 staining. DAPI counterstaining is presented to visualize cell nuclei. All images were acquired at identical magnification. Scale bars: 25 μm; 5 μm (insets). (C) Immunoblotting of cross-linked cell lysates demonstrated increased activation-associated trimeric (Tri) versus inactive monomeric (Mon) forms of HSF1 in MPNST cells without NF1 compared with lysates from NF1-expressing cell lines. The trimeric/monomeric ratio in lysates, as measured by densitometry, is indicated. (D and E) MEK inhibition reduced p-Ser326 and HSF1 nuclear translocation in human MPNST cells. S462 cells were treated with 20 μM U0126 overnight. Total p-Ser326 and nuclear HSF1 were detected by immunoblotting.