Figure 1. Interaction of ALMS1 with C2-NKCC2.

(A) Representation of C2-NKCC2 domain (pink) and unique peptides corresponding to ALMS1 (red) picked up by liquid chromatography–mass spectrometry in glutathione-s-transferase (GST) pull-down assay using GST-carboxyl-terminus of NKCC2 fusion protein (GST-C2-NKCC2) or GST alone (control) as baits in rat thick ascending limb (TAL) lysates. (B) Immunofluorescent labeling for NKCC2 (green) and ALMS1 (red) in paraffin embedded rat kidney sections, indicating expression and colocalization of NKCC2 and ALMS1 in rat TAL. CD, collecting duct; n = 3. Scale bars: 20 μm. (C) Representative Western blot for immunoprecipitation of ALMS1 with NKCC2 in NRK 52E cells transduced with EGFP or EGFP-NKKC2 adenovirus construct; n = 3. (D) Western blot representing GST pull down of full-length NKCC2 with GST-truncated carboxyl-terminus ALMS1 fusion protein B (GST-C-ALMS1 B) as bait in rat TAL lysates; n = 3. The lanes were run on the same gel but were noncontiguous and contrast changed on the supernatant fraction for visualization of discrete protein bands. (E) Recreated IPA of protein-to-protein interactions identified in GST pull-down assay with GST-carboxyl-terminus ALMS1 fusion proteins A/B (GST-C-ALMS1 A/B) in rat TAL lysates detected by liquid chromatography–mass spectrometry; n = 1. Solid and broken lines in black indicate direct and indirect protein interactions from IPA, respectively. Solid blue lines indicate protein interactions from mass spectrometry analysis of C-ALMS1 A/B pull-down protein fraction. RAC1, ras-related C3 botulinum toxin substrate 1; FLOT1, flotillin 1; ANXA2, annexin A2; PICALM, phosphatidyl inositol binding clathrin assembly protein; ARPC5L, actin related protein 2/3 complex subunit 5–like; RALA, ras like proto-oncogene A; ARF1, ADP ribosylation factor 1; ILK, integrin linked kinase; PHB, prohibitin; ACTN4, α-actinin 4; MYO5B, myosin 5b; GSN, gelsolin. Respective roles of these proteins in regulating endocytosis are defined in Supplemental Table 1.