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. 2012 May 14;287(27):23216–23226. doi: 10.1074/jbc.M111.335927

FIGURE 1.

FIGURE 1.

Interaction of ErbB4 with PIAS3. A, schematic structure of full-length ErbB4 JM-a CYT-2, and GST fusion proteins containing the whole ErbB4 intracellular domain of CYT-2-type (ICD2), ICD2 with C- (ΔC), or N-terminal (ΔN) deletion. TK, tyrosine kinase. B, GST fusion proteins (A) were pulled down with glutathione-Sepharose and analyzed by Western blotting (W) with anti-GST. C, GST-fusion proteins (A) were incubated with lysates of COS-7 cells transiently expressing FLAG-tagged PIAS proteins, STAT5A, or Myc-tagged Wwox. Pull-downs were analyzed by Western blotting with indicated antibodies. D, COS-7 cells were transfected with indicated constructs. Lysates were immunoprecipitated (IP) with anti-ErbB4 (HFR-1) followed by Western blotting with anti-FLAG to detect PIAS3. The membrane was reblotted with anti-ErbB4 (sc-283). Expression of PIAS3 was controlled by Western blotting with anti-FLAG. E, MCF-7 cells were transfected with the indicated control siRNA (Ambion) or siRNAs targeting PIAS3, stimulated for 15 min with 50 ng/ml NRG-1, and fixed. Complexes of ErbB4 and PIAS3 were visualized with anti-ErbB4 (HFR-1) and anti-PIAS3 (ab22856) antibodies using in situ PLA. Each red dot represents a single interaction. The nuclei were stained with DAPI (blue). F, quantification of PLA signals per cell. Signals were classified as cytosolic or nuclear on the basis of their colocalization with DAPI. Data of two independent experiments are shown (mean ± S.D.). *, p < 0.05. Densitometric data of PIAS3 protein levels relative to control knockdown as detected by Western blotting are indicated (protein (%)). G, COS-7 cells transfected with the indicated HA-tagged ErbB4 ICD2 and either His- or GFP-tagged SUMO-1 constructs were lysed in the presence of 20 mm N-ethylmaleimide. Lysates were analyzed by immunoprecipitation and Western blotting with anti-HA. H, COS-7 cells were transfected with HA-tagged ErbB4 ICD2 and the indicated FLAG-tagged PIAS3 and His-tagged SUMO-1 constructs. Lysates prepared as in G were immunoprecipitated with anti-ErbB4 (HFR-1) followed by Western blotting with anti-HA. Expression of PIAS3 was controlled by Western blotting with anti-FLAG. The band marked with an asterisk presumably represents another SUMO modification site.