Figure 6: Myo9A expression and Myo9A-actin-calmodulin interaction knock-in Myo9A^R701X kidneys and podocytes.

A) IF detects glomerular Myo9A and podocin: Myo9A glomerular immunostaining (red) is strong in wild type kidneys, decreases significantly in Myo9A^R701X/+ glomeruli, and is not detected in Myo9A^R701X/R701X glomeruli; podocin (green) localizes to podocytes in all mice; podocyte-Myo9A partial co-localization is shown by merge (yellow). B) Immunoblotting: Intact Myo9A (280kDa) expression decreases to ~ half in Myo9A^R701X/+ (Het) kidneys and is not detected in Myo9A^R701X/R701X (Hom) kidneys, smaller bands (<75kDa) are considered non-specific, ~50 kDa bands correspond to IgG heavy chain in all genotypes. C) Myo9A/actin quantification (mean±SD), n=3 independent experiments, pool from 3 mice each, ** indicates P<0.001. D) qPCR: Myo9A mRNA expression normalized to GAPDH mRNA, fold change (mean±SD), n=3 independent experiments, n=3 individual mice each, **** indicates P<0.0001. E-F) co-IP of kidney lysates (E) and primary podocytes (F) show decreased Myo9A-actin-calmodulin interaction in Myo9A^R701X/+ (Het) proportionally to the reduced Myo9A expression (input), note input of actin and calmodulin is equal in both genotypes.