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. 2006 Jul 31;103(32):11934–11939. doi: 10.1073/pnas.0510306103

Fig. 4.

Fig. 4.

PIPK Iγ preferentially associates with AP-2 bound to membranes or endocytic receptor sorting signals. (A) AP-2-PIPK Iγ complexes can be isolated preferentially from membrane fractions. Detergent extracts of HEK293 cells inducibly expressing PIPK Iγ were subjected to immunoprecipitation using anti-HA affinity tag antibodies. Aliquots of the material immunoprecipitated from total cell lysates or cytosolic or membrane fractions were analyzed by immunoblotting with antibodies against AP-2α, talin, or HA-PIPK Iγ. STD, detergent extracted rat brain lysates (15 μg of total protein). (B) PIPK Iγ associates with AP-2 bound to the cytoplasmic tail of the EGFR. GST-stonin 1(1–33), the GST-tagged cytoplasmic tail of the EGF receptor (EGFR), or GST were used for affinity purification from detergent-extracted rat brain lysates. Samples were analyzed by SDS/PAGE and immunoblotting for AP-2μ or PIPK Iγ. STD, 5% of the total amount of Triton X-100-extracted rat brain lysates added to the assay. (C) AP-2 coimmunoprecipitates with both EGFR and PIPK Iγ. Detergent extracts of Cos7 cells that had been mock-treated or transfected with plasmids encoding HA-PIPK Iγ and the EGFRs were subjected to immunoprecipitation using antibodies against AP-2α. Aliquots of the supernatant or pellet fractions were analyzed by immunoblotting for AP-2α, EGFR, or HA-PIPK Iγ.