IB and co-IP assays

GMCs and renal tissues were lysed with RIPA or NE-PER lysis buffer containing a phosphatase inhibitor. Equal amounts (40 μg/lane) of protein were subjected to SDS-PAGE. IB analysis was performed²⁸. β-actin was used as an internal control for protein loading, and the relative protein level in each group was calculated by comparison to that in the control group.

Co‐IP was performed to enrich the target protein complex^31–33. Briefly, a total of 400 μg of extracts prepared from GMCs, cell nuclear, and cytoplasmic compartments or renal tissues were mixed with 40 μl Protein A/G-Sepharose Beads in co-IP assay buffer, incubated for 2 h and centrifuged for 2 min. The recovered supernatant was incubated with the corresponding Abs (2 g, IgG isotype as a control) at 4 °C for 12 h. Then, 40 ml of Protein A/G-Sepharose Beads were added, and the incubation was continued for 2 h. Protein A/G-precipitated protein complexes were recovered by centrifugation, and the harvested beads were resuspended in 50 μl of 2× SDS-PAGE sample buffer and boiled for 5 min. The samples were analyzed by IB. A 40 mg aliquot of whole-cell extract (WCE) was used as an input control.