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. 2015 Oct 14;10(10):e0140417. doi: 10.1371/journal.pone.0140417

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© 2015 Coffey et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 5 — HKC-8 cells were transfected with HA-minimegalin and treated with only albumin (A) and insulin+albumin (A+I). Megalin was pulled down by immunoprecipitation with a HA antibody (Roche) and probed for AS160 (rabbit, Milipore). Mouse IgG is used as negative control. The protein expression in the pellet (P), supernatant (S) and input (I) were displayed. The pellet showed significant AS160 expression where as negative control did not display any protein expression indicating a robust interaction between AS160 and megalin. Insulin treatment caused a modest increase in this interaction. The same blot was stripped and reprobed by a HA antibody to show the efficiency of the pull-down experiments (A). Reciprocal immunoprecipitation was accomplished by transfecting the HKC-8 cells with Flag-AS160 and minimegalin plasmids. AS160 was pulled down by immunoprecipitation with a Flag antibody (Sigma) and the membrane was probed for megalin. It was confirmed that megalin has a strong interaction with AS160. Insulin treatment did not alter the degree of interaction between AS160 and megalin. Pull-down with mouse IgG was utilized as a negative control. The same membrane was stripped and reprobed by a Flag antibody (the image at the lower panel) to examine the efficiency of pull-down experiment (B). Both experiments revealed a strong interaction between megalin and AS160. Megalin and AS160 were successfully pulled down by tag antibodies.