Arly, bandB was severely decreased though bandA remained (Fig 2B, left). Western blot working with an antiFLAG antibody revealed that bandA contained FLAG and was hence the SPuncleaved, immature ZIP13 protein (Fig 2B, middle). BandB was recognized in the FWT sample by abA1 (Fig 2B, correct), but not by the antiFLAG antibody (Fig 2B, middle), indicating that it was the SPcleaved, mature ZIP13WT protein. No bands had been detected by the abA1 antibody within the FG64D sample (Fig 2B, ideal), indicating that the SPcleaved ZIP13G64D mature protein was especially decreased inside the cells. Western blot using the abA2 antibody revealed bandB at a reduced position, most likely corresponding for the SPcleaved, mature ZIP13 protein (Fig 2C, middle), as well as the volume of bandB yielded by the expression plasmid for FG64D was markedly decreased (Fig 2C, middle). Furthermore, when the lysates from cells expressing a Cterminally V5 epitopetagged ZIP13 (ZIP13V5) (Fig 2D) have been subjected to Western blot with an antiV5 antibody, the V5tagged mutant (G64DV5) levels have been lower (Fig 2E and Supplementary Fig S2A), equivalent towards the final results with FG64D (Fig 2B). Although immunoprecipitation evaluation showed the exact same two bands in each the wildtype (WTV5) and G64DV5 samples (Fig 2E, bandA and bandB), the2014 The AuthorsEMBO Molecular Medicine Vol six | No eight |EMBO Molecular MedicinePathogenic mechanism by ZIP13 mutantsBumHo Bin et alABNLumen CMT1 mRNA expression ( of handle)4 3 two 1G64DMockpZIP13G64DpZIP13WTplasmid:CytosolpZIP13G64D pZIP13WTC DMockplasmid:SPC cleavage siteG64 ZIP13 SPZIP13 GAPDHabAabAEplasmid: ( g)pZIP13WT 0 five ten 20pZIP13G64D 5 10IB: abAIB: TUBULINFigure 1. ZIP13 with the pathogenic G64D mutation shows a decreased protein expression level. A Place on the G64D mutation in ZIP13. Asterisk () indicates the G64D mutation. B Metallothionein 1 (MT1) expression. 293T cells transfected using the indicated DNA constructs were treated with 50 lM ZnSO4 for six h, and then, the MT1 mRNA expression level was analyzed by RTqPCR. Information are representative of 3 experiments and shown as imply s.e.m. (P = 0.037). ZIP14WT was included as a constructive handle. C ZIP13 transcript levels in 293T cells expressing wildtype or G64D mutant ZIP13.4-Chloro-5-methoxypyridin-2-amine uses 293T cells were transfected with plasmids for ZIP13WT or ZIP13G64D.Formula of 2306261-01-6 Twentyfour hours later, RT CR was performed using primers for the indicated genes (Fukada et al, 2008).PMID:24516446 D Schematic diagram displaying the recognition web pages of antiZIP13 antibodies. Asterisk () indicates the G64D mutation. SP, signal peptide; SPC, signal peptidase complicated; abA1 and abA2 indicate antiZIP13 antibodies that recognize amino acids 235 of human ZIP13 and 18401 of mouse ZIP13, respectively. E ZIP13 protein levels in 293T cells expressing wildtype and G64D mutant ZIP13. Cell lysates were analyzed by Western blot (IB) applying the abA1 antibody. Supply information are out there on-line for this figure.G64DV5expressing cells contained a reduced amount of bandB, indicating that the expression of SPcleaved G64D mature protein was tremendously decreased in these cells. Given that ZIP13 protein forms a homodimer (Bin et al, 2011) and the G87R mutation within the zinc transporter ZnT2 is reported to result in neonatal zinc deficiency due to a dominantnegative impact on its homodimerization (Lasry et al, 2012), we next examined whether or not the G64D mutation affects the oligomeric state from the ZIP13 protein. Blue nativePAGE analysis of lysates from FZIP13expressing 293T cells showed a lower expression of FG64D than FWT, however the FG.
