Western blotting of wild-type and variant PC1/3 proteins expressed in HEK cells.

<p>HEK cells were transiently transfected with empty pcDNA3 (E); pcDNA3 encoding either wild-type PC1/3; or PC1/3 proteins bearing the mutations under study. Western blots of cell lysates and media from transfected HEK cells were probed with amino-terminally directed PC1/3 primary antiserum for detection of recombinant proteins. The data shown represent 1 of 3 independent experiments performed in triplicate. Total secreted immunoreactive band intensity values, obtained through densitometry analysis and used to calculate specific activity for each variant, are represented above the Western blot and shown as the mean ± S.D.</p

Western blotting of wild-type and novel R80Q (rs1799904) variant PC1/3s, expressed in Neuro-2A cells.

<p><b>Panel A:</b> Neuro-2a cells were transiently transfected with equal amounts of empty pcDNA3 (E), or pcDNA3 encoding wild-type PC1/3 or the novel variant R80Q (rs1799904) PC1/3. Western blots of media were probed using amino-terminally directed PC1/3 primary antiserum. The data shown represent one of 3 independent experiments performed in triplicate. <b>Panel B: Specific activities of wild-type PC1/3 and the R80Q PC1/3 variant.</b> Enzymatic activities of secreted recombinant PC1/3 proteins in conditioned medium were compared by measuring maximum cleavage rates using the fluorogenic substrate pyr-ERTKR-amc during a 1 h kinetic assay. Three replicates per transfection condition were assayed in triplicate, and maximum rates were divided by band intensity of immunoreactive protein in the spent medium of the same wells from which the activity data were derived. Specific activity values are shown as the mean ± S.D. Data represent one of 3 independent experiments, each performed in triplicate.</p

Potentially consequential variant alleles in <i>PCSK1</i>.

<p>The R80Q (rs1799904) variant that differed from the human reference genome and was predicted to have a potentially consequential effect on the transcript was selected from the dbSNP 137, 1000 Genomes, NHLBI, and NIEHS public datasets. <i>Pos</i>: genomic position in GRCh37; <i>ID</i>: dbSNP 137 rs ID; <i>REF</i>: reference allele; <i>ALT</i>: alternate allele (variant); <i>Rank</i>: 3 splice_donor_variant, 12 missense_variant; <i>cDNA</i>: position and consequence of variant in cDNA of canonical NM_000439.4 transcript; <i>Protein</i>: position and consequence of variant in NP_000430.3 protein; <i>Effect</i>: computational prediction of effect on protein structure or function (“S” predicted deleterious by SIFT, “P” or “p” predicted probably or possibly damaging by PolyPhen, “C”, predicted deleterious by Condel from a consensus of SIFT and PolyPhen, “-” no prediction); <i>MAF</i>: minor allele frequency across all populations; <i>Samples</i>: total number of individuals genotyped; <i>Het</i>: number of individuals heterozygous for the variant allele; <i>Hom</i>: number of individuals homozygous for the variant allele. Known, common variants are listed in italics, and the rare novel variant is shown in bold.</p