FIGURE 6:

A CRH motif localizes KS-WNK1 to WNK bodies. (A) Amino acid sequence of exon 4a. The sequence emerged in coelacanths and is highly conserved to humans, including a putative CRH motif (highlighted in blue). (B) Exon 4a mutagenesis. Five constructs were created including 1) Δ30, lacking the entire exon 4a, 2) C6→S, mutating all of the cysteines to serines, 3) C2→S, mutating the two outer cysteines to serines, 4) C4→S, mutating the four inner cysteines to serines, and 5) 5Φ→5Q, mutating the hydrophobic cluster to glutamines. (C, D) SP assay (anti-HA immunoblot) of HEK-293 cells transiently transfected with KS-WNK1-HA mutants. Representative immunofluorescence of HEK-293 cells transiently transfected with either KS-WNK1-HA or N-terminal mutants, probed with HA antibody (n = 3 transfections). (E) Fluorescence recovery after photobleaching (FRAP) experiment. HEK-293 cells were transiently transfected with L-WNK1-GFP, KS-WNK1-GFP, or C6→S-KS-WNK1-GFP. Representative time series of WNK1-GFP recovery after photobleaching (area bleached circled in blue and recovery indicated by yellow arrows). (F) Percent recovery of the mobile fraction over 20 s. L-WNK1 and the C6→S mutant exhibited a similar rate of recovery (n = 10 replicates for both). In contrast, KS-WNK1 puncta (n = 3 replicates) exhibited limited mobility, with only 5% recovery by 20 s. Error bars = SEM.