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. 2004 Oct 26;383(Pt 3):447–455. doi: 10.1042/BJ20040498

Figure 11. Time courses of JNK and MKK4 activation in the presence or absence of LDP-3 upon sorbitol stimulation.

Figure 11

COS-7 cells were co-transfected with 0.5 μg of pSRα-HA-JNK1 (A) or pcDNA-HA-MKK4KR (B) together with 1.5 μg of pFLAG-CMV or pFLAG-LDP-3. After 36 h of culture, cells were treated with 0.4 M sorbitol and incubated for the indicated times. Phosphorylated HA–JNK1 and HA–MKK4KR were detected by anti-ACTIVE JNK and phospho-SEK1/MKK4 antibodies respectively (SEK is stress-activated protein kinase/ERK kinase). The levels of expression of HA–JNK1 and HA–MKK4KR were determined by immunoblot with anti-HA antibody. Expression levels of FLAG–LDP-3 were assessed by immunoblot using anti-FLAG antibody. Signals were detected with a Fuji LAS 1000 image analyser and quantified. The graph shows the relative intensity of phosphorylated JNK and MKK4 in the absence (○) or presence (•) of LDP-3, at various time points. Intensities relative to that seen in cells stimulated with 0.4 M sorbitol for 30 min without forced expression of FLAG–LDP-3 are presented. Data are representative of three independent experiments.