Fig. 3: Na-pump recruitment to zDHHC5 can be regulated by palmitoylation.

a HEK 293 cells were transiently transfected to express WT DHHC5 or catalytically inactive DHHS5 +/− C236A/C237A, (AA), C245A, (A) or C236A/C237A/C245A, (AAA) mutants. Palmitoylated fractions were purified by resin-assisted capture (Acyl-RAC) and visualized by Western Blot. The average data is shown in graph form with palmitoylation expressed as the ratio of Palmitoylated (Palm)/Unfractionated (UF) zDHHC/S5 (n = 3: *p < 0.05, **p < 0.01). b The number of sites palmitoylated in zDHHC5, zDHHS5, and three zDHHS5 C-tail mutants (236/7AA, 245A and 236/7–245AAA) was determined by Acyl PEG exchange. c Co-immunoprecipitation of HA-tagged zDHHC5 and 236/7AA mutant with PLM. The amount of PLM co-immunoprecipitated was expressed relative to the amount of zDHHC5 immunoprecipitated (n = 3: ****p < 0.0001). d HEK 293 cells were engineered with CRISPR/Cas9 to create a zDHHC5 knockout cell line (zDHHC5 KO). zDHHC5 can be reintroduced to the KO cells by transient transfection. e Both HEK 293 and zDHHC5 KO cells were transiently transfected to express zDHHS5. The bar chart shows relative palmitoylation (Palm/UF) of zDHHS5 as determined by Acyl-RAC (n = 3). f zDHHS5 and C236/7AA-zDHHS5 were expressed in HEK cells +/− zDHHC20. The palmitoylation status (Palm/UF) of each zDHHS5 mutant was determined by Acyl-RAC. (n = 4: *p < 0.05, **p < 0.01). g Impact of wild type and catalytically inactive zDHHC20 on the palmitoylation status of zDHHS5 (n = 4: *p < 0.05, **p < 0.01). h HA-tagged WT zDHHC5 and the C236/7AA mutant were expressed in HEK 293 cells +/− zDHHC20 and immunoprecipitated with HA beads. Associated PLM was visualized by Western Blotting.