Figure 2

Puromycin treated podocytes show increased levels of L1-32 and soluble L1. (A) Human podocytes were treated for 24 h with 5 μg/ml and 10 μg/ml puromycin. Cells were lysed and western blot experiments were done with an antibody against the cytoplasmic tail of L1. (B) Human podocytes were treated for 6 h and 24 h with 5 μg/ml and 10 μg/ml puromycin (Puro), supernatants were collected and after TCA-precipitation, equal amounts of protein samples were loaded on a SDS-PAGE. Membranes were probed with L1-11A, an antibody against the ectodomain of L1. (C) Human podocytes were pretreated 30 min with 3 μM ADAM10 inhibitor GI254023X (GI) before incubating cells for 6 hours with 10 μg/ml puromycin (Puro). Supernatants were analyzed for soluble L1 by western blot analysis. (D) Western Blot analysis of soluble L1 after the transfection of ADAM10 specific siRNA in the presence or absence of 5 μg/ml puromycin (24 hour treatment). As a negative control a scrambled siRNA was used (A10 = ADAM10, sc = Scrambled, Puro = Puromycin). Efficient knockdown of ADAM10 was controlled by westernblot with ADAM10 specific antibody (A10 = ADAM10, sc = scrambled) and equal loading of the samples were determined by β-actin westernblot.