Pepmute sirna transfection reagent

The transient transfection assay was carried out in 6-well plates using PepMute™ siRNA Transfection Reagent (SignaGen® Laboratories, USA) according to the manufacturer's instruction. First, PepMute™ transfection buffer was diluted before use, and then 3.3 μl siRNA in 1 ml RPMI 1640 medium was added gently and incubated for 15 min at room temperature. The complexes were added to each well and mixed gently by rocking the plates back and forth. The plates were then incubated at 37°C in a CO₂ incubator. After 48 h, cells were collected for further experiments. The CXCR4 siRNA product was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

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)].

For gene knockdown experiments, c-Myc siRNA and non-targeting scrambled siRNA (NS) obtained from Santa Cruz Biotech were transfected into RPMI 8226 cells using PepMute™ siRNA transfection reagent (SignaGen Laboratories). For gene overexpression experiments, cells were transfected with pcDNA3.1-c-Myc, Flag-SUMO1 or HA-Ubiquitin plasmids (Addgene) using GenJet™ in vitro DNA transfection reagent (SignaGen Laboratories).