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. 2012 Jul 1;26(13):1421–1426. doi: 10.1101/gad.190876.112

Figure 4.

Figure 4.

NF1 is necessary for Spred1's inhibitory function. (A) Neurofibromin was siRNA-depleted from TET-inducible 6X-HIS eGFPf and Spred1 cell lines, serum-starved, and stimulated with EGF for the times indicated. Active Ras was precipitated from whole-cell lysates using GST-Raf RBD beads. (B) MEFs were transduced with eGFPf- or Spred1-expressing retrovirus. Following serum starvation, cells were treated with EGF for the indicated times, lysed, and analyzed by immunoblot. (C) HeLa cells were transfected with either GFP-NF1 alone, myc-Spred1 alone, GFP-NF1 and myc-Spred1, or GFP-NF1 and the indicated myc-Spred1 mutant and serum-starved and for 16 h. Fixed cells were immunostained for myc (red), GFP (green), and DNA (DAPI). (D,E) HEK-293T cells ectopically expressing either wild-type Spred1 or the indicated Spred1 mutant (D) or Spred2 (E) were subjected to biochemical fractionation, and the resulting fractions were resolved by immunoblot analysis.