Lipofectamine ranimax

Cells were transfected with Lipofectamine RANi MAX (Invitrogen) in culture medium without antibiotics. Silencer Select Pre-designed siRNAs (Ambion, Loughborough, UK) against moesin (ID s70074), radixin (ID s201923), annexin II (ID s63242), fodrin (ID s232645), and Silencer Select Negative Control siRNA (#1) were used at a final concentration of 5 nM. The knockdown and cell phenotype were assessed 24, 48, and 72 h later.
Cells were grown on numbered grids, transfected with siRNA, and induced to form fenestrae using the standard protocol. Cells were fixed with 4% PFA/0.1% glutaraldehyde for 15 min and then were immunolabelled. Cells with no detectable immunostaining for the target protein were identified under an epifluorescence microscope, and their position was recorded. After processing for TEM, the identified cells were re-located under electron microscopy and imaged as described in Section 2.6.

Knockdown of WNK1 or OSR1 protein expression was induced by small interfering RNA (siRNA). The scramble siRNA (Silencer^® Negative Control No. 1 siRNA, Cat. No. AM4635) and siRNAs targeting human WNK1 (ID: s35235) and OSR1 (ID: s19302) were purchased from Invitrogen. The sequences of WNK1 siRNAs are: sense 5′-CAAUGSGUCAGAUAUACGAAtt-3′; antisense 5′-UUCGAUAUCUGACUCAUUGtc-3′; OSR1 siRNA: sense 5′-GAACCUCAGUCAAAUCGAUtt-3′; antisense 5′-AUCGAUUUGACUGAGGUUCtt-3′. Dissociated GCs were seeded in 6-well plates (10⁵ cells/well/2 ml) in DMEM plus 10% FBS at 24 h prior to transfection. Lipofectamine RNAiMAX/siRNA complexes were prepared by adding siRNA (15 nM as a final concentration) and 5 μl of Lipofectamine RANiMAX (Invitrogen, Carlsbad, CA) in 500 μL serum-free optiMEM. Complexes were allowed to form at RT for 10 min and added to each well in the 6-well plate. The cells were incubated at 37°C and subjected to experiments 48 h after transfection.