Non targeted control sirna

Specific siRNAs targeting mouse calpain 1 and a non-targeted control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Podocytes were seeded in a 6-well culture dish, and siRNA duplexes were diluted in 100 μl of serum-free medium. Then, 6 μl of Lipofectamine 2000 (Invitrogen, USA) was added to 100 μl of the siRNA duplexes. The duplexes were incubated for 15 min and subsequently added to each well. After 48 h of transfection, the cells were treated using the appropriate methods. After the cells were lysed in RIPA, Western blots were performed to examine protein levels.

Specific siRNAs targeting mouse raptor and rictor (each siRNA product consists of a pool of 3 target-specific 19–25 nt siRNAs designed to knock down the gene expression) and non-targeted control siRNA were purchased from Santa Cruz (Santa Cruz, USA). Podocytes were seeded on a 6-well culture dish or coverslips on the preceding day to reach 50–60% confluence at the time of transfection. For each well, siRNA duplexes were diluted into 100 µl of serum-free medium. Then, 6 µl of lipofectamine 2000 (Invitrogen, USA) was added to 100 µl of the siRNA duplexes. The duplexes were incubated for 15 minutes and added to each well. After 48 hours of transfection, the cells were lysed for Western blotting or were used for calcium imaging.