Fig. 1.

Nephrin binds to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel subunits. A: coimmunoprecipitation of nephrin observed by immunoblot analysis after immunoprecipitation using antibodies against Slo1 or the β4-subunit of BK_Ca channels from mouse kidney or mouse brain, as indicated. The “input” lane contained a diluted sample of cell extract used to illustrate electrophoretic mobility; it was not designed for quantification of signal intensity. B: coimmunoprecipitation of Slo1 observed by immunoblot analysis after immunoprecipitation with an antibody against nephrin (top). This procedure was carried out on HEK-293T cells transiently expressing Myc-tagged isoforms Slo1_VEDEC, Slo1_QEERL, or Slo1_EMVYR, as indicated, and channels were detected using anti-Myc. Note that a strong signal was only detected from cells expressing the Slo1_VEDEC isoform. Interaction with Slo1_EMVYR was detectable but much weaker. IB analysis shows that the various Slo1 variants (middle) and nephrin (bottom) were expressed at comparable levels throughout the assay. C: glutathione S-transferase (GST) pull-down assay showing that a GST fusion protein containing the COOH-terminal sequences of Slo1_VEDEC could bind to nephrin, whereas GST and a GST fusion protein containing the COOH-terminal sequences of Slo1_QEERL did not. The interaction with Slo1_EMVYR with nephrin was very weak. D: confocal microscopy showing colocalization of endogenous Slo1 and nephrin in differentiated cells of a podocyte cell line (top) and in HEK-293T cells transiently expressing nephrin and Slo1_VEDEC (bottom). Middle, control images from podocytes in which the primary antibody was a species-appropriate IgG. E: immunoblot using the same commercial antibody against nephrin on extracts of differentiated and undifferentiated cells from a podocyte cell line. IB, immunoblot; IP, immunoprecipitation.