Fig. 4. Analysis of downstream signaling. (A) Serum-starved SK-N-SH cells were stimulated with 4 nM anti-ALK mAb16-39 for 5 minutes. Lysates were immunoprecipitated (IP) with anti-ALK (ALK) or anti-Shc (Shc) antibodies or were immunoprecipitated without antibodies (mock) and immunoblotted with anti-PY antibody (upper panels). The same blots were reprobed with anti-ALK (middle panels) or anti-Shc (lower panels) antibodies. The positions of ALK (220 and 140 kDa) and three isoforms of Shc (66, 52, and 44 kDa) are indicated by the arrowheads and bracket, respectively. The dot shows the position of Shc (66 kDa). (B,C,D,E) Lysates prepared as above were immunoprecipitated with antibodies as indicated and immunoblotted with anti-PY antibody (upper panels) and then reprobed with the indicated antibodies (lower panels). The positions of the immunoprecipitated proteins are indicated by dots. Asterisks in C show unidentified co-immunoprecipitated proteins with anti-p85 antibody.