Fig 6. Residual nephrin at the membrane is associated with actin.
(A) Isolated glomeruli from wild type and Nphs1Tam-Cre mice kidneys were lysed in RIPA buffer containing 1% Triton X-100 at 4°C. Lysates were resolved with SDS-PAGE gel under reducing conditions and probed with nephrin and actin antibodies. Triton X1000 soluble fraction (S) and insoluble pellet (P) were separated by centrifugation. Nephrin is seen primarily in the insoluble fraction or pellet. (B) Glomerular lysates in RIPA buffer at 37°C showing no improvement in nephrin solubulity. (C) Glomerular lysates in RIPA buffer without 1.5 M MgCl2 shows slight improvement in nephrin solubility with prominence of the high molecular weight band in the Nphs1Tam-Cre mice. (D) Glomerular lysates in RIPA buffer containing 1M potassium iodide (KI) show dramatic improvement in nephrin solubility. IB: Immunoblot.