Figure 11.

Co‐immunoprecipitation (Co‐IP) experiments. A, Co‐IPs were performed with various antibodies against α‐subunits of different channels to examine the possible associations between XIRP and channel components. Total lysates (lane 1) and clarified supernatants as inputs (lane 2) were prepared from adult wild‐type mouse hearts as described under Methods. The aliquot of input was used for immunoprecipitation (IP) with various primary antibodies cross‐linked to Dynabeads protein G. The primary antibodies included rabbit anti‐Kv1.5 (lane 3), rabbit anti‐Nav1.5 (lane 4), mouse anti‐Kv4.2 (lane 5), and rabbit anti‐GAPDH (lane 6). The resulting immunoprecipitates were fractionated and immunoblotted with rabbit anti‐Xirp1 (U1013) antibody that recognized both Xirp1‐S (small isoform), Xirp‐L (large isoform), and Xirp2. Xirp2 was co‐pelleted in the immunoprecipitates with anti‐Kv1.5 and with anti‐Nav1.5, but neither with anti‐Kv4.2 nor with anti‐GAPDH. B, Reverse Co‐IPs were performed with cross‐linked rabbit antibody U1697 (specific to Xirp1), U1040 (specific to Xirp2), U1013 (cross‐reacted to both Xirp1 and Xirp2), or anti‐GAPDH antibody. The resulting immunoprecipitates were immunoblotted with anti‐Kv1.5 (lower panel), anti‐Nav1.5 (top panel), or anti‐Kv4.2 (data not shown). Both Kv1.5 and Nav1.5 are co‐pelleted in the immunoprecipitates with anti‐Xirp2 U1040 antibody.