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Fig. 2. Monoclonal anti-ALK antibody 16-39 induces tyrosine phosphorylation of ALK. Untransfected NIH 3T3 cells (NIH), derivative cells expressing mouse ALK (2-C4), or human neuroblastoma cells SK-N-SH (SK) were treated with 20 nM normal rat IgG (–) or mAb16-39 (+) for 5 minutes at 37°C. Lysates were immunoprecipitated with polyclonal anti-ALK antibodies that recognize the carboxyl terminus of ALK. The phosphotyrosine level was examined by western blotting (Blot) with anti-PY antibody RC20-HRP (upper panels). The presence of equal amounts of ALK was confirmed by reprobing with polyclonal anti-ALK antibodies (lower panels). Arrowheads indicate the positions of endogenous and transfected ALK (220, 140, and 85 kDa in 2-C4 cells and 220 and 140 kDa in SK-N-SH cells). The bracket shows the position of phosphoproteins of 66, 52, and 46 kDa in both 2-C4 and SK-N-SH cells. Asterisks indicate phosphoproteins of 160, 120, 85, and 35 kDa in SK-N-SH cells.
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