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. 2004 May 27;23(12):2358–2368. doi: 10.1038/sj.emboj.7600253

Figure 4.

Figure 4

ARMS4.2 interferes with sustained MAPK phosphorylation. (A) Neurite outgrowth of PC12 cells transfected with GFP, FLAG-ARMS or FLAG-ARMS4.2. Cells were stimulated with NGF for 24 h after transfection and neurite outgrowth was quantified 3 days later by assessing the percentage of fluorescence-positive cells bearing neurites at least twice the length of the cell body. At least 50 cells were counted for each experiment and condition. Results were normalized to control GFP-transfected cells (set as 100%). Values are calculated from at least four independent experiments. Mean and standard deviation (s.d.) are shown. Where applicable, statistical analyses were carried out by Student's t-test (*P<0.02; **P<0.001). (B) Activation of p-MAPK in PC12 cells transfected with GFP, FLAG-ARMS or FLAG-ARMS4.2. Respective cultures were stimulated with NGF for 5 or 40 min or EGF for 5 min. Quantification was carried out as described in panel A (**P<0.001). (C) Immunofluorescence analysis of primary cortical neurons transfected with ARMS4.2 constructs. Cells were transfected with GFP or GFP/FLAG-ARMS4.2 and stimulated 2 days later with BDNF for 40 min. Panel b shows the absence of p-MAPK staining in untreated cells. Panels d–l show transfected neurons with GFP and GFP/FLAG-ARMS4.2. Panels d–f and g–l show GFP and GFP/FLAG-ARMS4.2-transfected neurons that are positive (yellow, panel f) and negative (green, panel i) for p-MAPK, respectively. Panels j–l show GFP/FLAG-ARMS4.2-transfected neurons stained for p-Akt (yellow, panel l). Scale bar=5 μm. (D) Activation of p-MAPK in cortical neurons transfected with GFP and GFP/ARMS4.2 stimulated with BDNF for 40 min (**P<0.001).