Modulating Podocyte Cdk5 and p35 Pathways

Cdk5 siRNA (sc-29263), p35 siRNA (sc-36154), and control siRNA (sc-37007) were ordered from Santa Cruz Inc and delivered to podocytes using Pepmute siRNA transfection reagent (SignaGen Laboratories), according to the manufacturer's instructions. Briefly, podocytes were seeded in a 6-well plate and cultured for 7 days at about 50–60% confluence. For transfection, 5μl siRNA were diluted in 100μl of 1x siRNA transfection buffer (SignaGen Laboratories) in a final concentration of 50 nM siRNA. Three ul of Pepmute reagent were then mixed by pipetting up and down, incubated 15 minutes at RT, and dropped onto the cultured cells. After 72 h, cells were harvested or fixed for further experiments.
Adenovirus-p35 and empty vector (EV) were made and infected according to the methods of previous study [2 (link), 18 (link)].