Protein a or g agarose sepharose beads

Kidney cortex homogenate protein samples (1 mg), dissected proximal tubular segments (40 μg), or homogenate protein samples from murine smooth muscle cells (1 mg) were incubated with 2 μg of WNK4 (Novus Biologicals), TRPV5 (Lifespan Biosciences), anti-phosphoserine (Alpha Diagnostics), or anti-NaPi-2c (kind gift of Drs. Murer and Biber) antibody at 4°C overnight. The immune complexes were captured by adding 50 μl Protein A or G agarose/sepharose beads (Santa Cruz Biotechnology) and overnight incubation at 4°C with gentle rocking. The immunoprecipitates were collected by centrifugation at 1,000 g for 5 min at 4°C and washed for four times in PBS, each time repeating the centrifugation step. After the final wash, the pellets were suspended in 40 μl of electrophoresis sample buffer and boiled for 2–3 min. Western blot analysis was performed using a primary anti-TRPV5 or anti-WNK4 antibody. Each experiment was performed in duplicates and included 4–8 animals per group.

Kidney cortex homogenate protein samples (1 mg) were incubated with 2 μg of anti-WNK4 (Novus Biologicals), anti-phosphoserine (Alpha Diagnostics), or anti-NCC (Millipore) antibody at 4°C overnight. The immune complexes were captured by adding 50 μl Protein A or G agarose/sepharose beads (Santa Cruz Biotechnology) and overnight incubation at 4°C with gentle rocking. The immunoprecipitates were collected by centrifugation at 1,000 × g for 5 min at 4°C and washed for four times in PBS, each time repeating the centrifugation step. After the final wash, the pellets were suspended in 40 μl of electrophoresis sample buffer and boiled for 2–3 min. Western blot analysis was performed as described above using a primary anti-NCC, anti-phosphoserine, or anti-WNK4 antibody.