Figure 6

Podocyte RT-PCR expression analysis of Mag. Mouse kidney and two mouse podocyte cell lines (K5 D and S6) were cultured as reported earlier [63]. Podocyte RNA was isolated using a mixture of guanidine thiocyanate and phenol (TRI Reagent, Sigma) according to the manufacturer's protocol. Reverse transcription was performed on 5 μ g denaturated RNA. Real-time PCR was performed with a Light Cycler (Roche Diagnostics, Mannheim, Germany) using the Platinum^® SYBR^® Green qPCR SuperMix-UDG kit (Invitrogen, Heidelberg, Germany) and run at 95°C for 10 min followed by 45 cycles at 95°C for 10 s, 60°C for 5 s, and 72°C for 12 s. Relative expression was normalized using GAPDH. The following primers were used: Mag sense 5'-TGG GCC TAC GAA ACT GTA CC-3', anti-sense: 5'-GCT CCG AGA AGG TGT ACT GG-3', 110 bp expected product size; Gapdh sense 5'-ACC CAG AAG ACT GTG GAT GG-3', antisense 5'-CAC ATT GGG GGT AGG AAC AC-3', 170 bp expected product size. A 50 bp DNA step ladder was loaded in the left lanes.