Journal: The Journal of Biological Chemistry

Article Title: Palmitoylation of Progressive Rod-Cone Degeneration (PRCD) Regulates Protein Stability and Localization *

doi: 10.1074/jbc.M116.742767

Figure Lengend Snippet: The gBlocks fragments of human PRCD wild type and mutant gene sequences were synthesized from Integrated DNA Technologies used for cloning into pGAG-EGFP vector for cell culture and subretinal injection studies The restriction sites for cloning in to pGAG-EGFP vector are underlined, and the bolded sequences in the 3′ sites represent the HA epitope tag. The changes made in the sequence according to human mutations are underlined and bolded.

Article Snippet: The human PRCD constructs encoding wild type and mutants (C2Y and V30M) with the C-terminal HA epitope-tagged gBlocks gene fragments (200 ng) were custom-synthesized from Integrated DNA Technologies.

Techniques: Mutagenesis, Synthesized, Clone Assay, Plasmid Preparation, Cell Culture, Injection, Sequencing

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: Deep sequencing of the ST18 gene locus in 16 PV patients led to identification of 789 genetic variants (black dots), depicted along the ST18 gene locus (X axis, chr8:53,023,392–53,373,519, GRCh37/hg19 assembly; Y axis, negative log-transformed P-values of association score). A case-control association analysis against the 1000 genome project data revealed a large PV-associated haplotype block (in red) residing within an ST18 intron and harboring rs17315309 (arrows).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Sequencing, Transformation Assay, Control, Blocking Assay

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: (a) rs17315309 resides within a p53/p63 half binding site, upstream to the ST18 coding sequence, and results in the substitution of a highly conserved T nucleotide (broken arrow) within the consensus binding sites of p53 and p63 (MotifMap database; http://motifmap.ics.uci.edu/ ); (b) NHEKs were co-transfected with a luciferase reporter construct under the regulation of a 282 bp fragment from the putative ST18 promoter, harboring either the wild type or the risk allele of rs17315309, and with control (si-Cont), TP53- (si-p53) or TP63- (si-p63) specific siRNAs. Luciferase activity measurements are provided as arbitrary units (a.u.) relative to the luciferase activity measured in cells transfected with wild type allele and control siRNA. Results represent the mean of three independent experiments ± SE (*p<0.05, **p<0.01 and ***p<0.001 by 2-tailed t test).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Construct, Control, Activity Assay

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: NHEKs were transfected with an ST18 expression vector (ST18, dark grey) or control empty vector (EV, light grey). (a,b,c) Supernatants were collected 2, 4, 6 and 24 hours post exposure to PV serum or control serum and (a) TNFα, (b) IL-1α and (c) IL-6 secretion was measured as described in Materials and Methods; (d,e,f) Supernatants were collected 6 hours (IL-1α and IL-6) or 24 hours (TNFα) post exposure to PV and control serum or PV and control IgG and (d) TNFα, (e) IL-1α and (f) IL-6 secretion was measured as described in Materials and Methods. Results represent the mean of three independent experiments and are expressed as the relative cytokine secretion in percentage, compared to control (empty vector) ± SE (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by 2-tailed t test).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Transfection, Expressing, Plasmid Preparation, Control

Journal: PLoS Genetics

Article Title: Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

doi: 10.1371/journal.pgen.1006008

Figure Lengend Snippet: We used a dispase-based dissociation assay to evaluate the effect of ST18 overexpression on cell adhesion. NHEKs transfected with a ST18 expression vector (ST18) or with a control vector (EV) were grown to confluency in the presence of PV serum and control serum or PV IgG and control IgG. (a) Epidermal sheets were released from the tissue plates and subjected to mechanical stress as described in Materials and Methods; (b) the resulting fragments were counted. Results represent the mean of three independent experiments and are expressed as number of fragments ± SE (*p<0.05, **p<0.01, ***p<0.001 by 2-tailed t test).

Article Snippet: A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively.

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Control