anti islr2  (Merck & Co)

Journal: Human Molecular Genetics

Article Title: The protective variant rs7173049 at LOXL1 locus impacts on retinoic acid signaling pathway in pseudoexfoliation syndrome

doi: 10.1093/hmg/ddz075

Figure Lengend Snippet: Transcriptional regulation of LOXL1 , STRA6 and ISLR2 by genomic region containing rs7173049. ( A ) Schematic diagram showing the genomic region 1 Mb up- and downstream of rs7173049 (chr15:73244610-75244610) containing 31 genes. ( B ) Quantitative real-time PCR assays of all 31 neighboring genes showing significant downregulation of LOXL1 as well as significant upregulation of STRA6 and ISLR2 in HEK293T cells after deletion of a 200 bp region flanking rs7173049 using CRISPR/Cas9 technology. Data represent mean values of eight edited (Deletion) and six non-edited (Control) clones; relative expression levels were normalized to GAPDH and are represented as means ± SD ( * P < 0.05; ** P < 0.01). ( C ) Immunofluorescence staining of HEK293T cells confirming reduced protein expression of LOXL1 as well as increased protein expression of STRA6 and ISLR2 in genome-edited cells compared to non-edited control cells. Nuclei are counterstained with DAPI (blue); scale bars are equal to 25 μ m .

Article Snippet: Cryosections of 4 μ m thickness were fixed in cold acetone for 10 min, washed in phosphate balanced saline (PBS) and permeabilized using 0.1% Triton X-100 in PBS for 10 min. After blocking with 10% normal goat serum, sections were incubated over night at 4°C in primary antibodies against STRA6 (ab169600, Abcam, Cambridge, UK), ISLR2 (ab81254, Abcam) and LOXL1 (kindly provided by Takako Sasaki, Department of Biochemistry II, Oita University, Japan) diluted in PBS.

Techniques: Real-time Polymerase Chain Reaction, CRISPR, Clone Assay, Expressing, Immunofluorescence, Staining

Journal: Human Molecular Genetics

Article Title: The protective variant rs7173049 at LOXL1 locus impacts on retinoic acid signaling pathway in pseudoexfoliation syndrome

doi: 10.1093/hmg/ddz075

Figure Lengend Snippet: Correlation of rs7173049 genotype and tissue expression levels of STRA6 and ISLR2. ( A ) Genotype-correlated mRNA expression levels of STRA6 and ISLR2 in human iris tissue samples homozygous for allele A ( n = 33) or allele G ( n = 5) or heterozygous for both alleles ( n = 28) at rs7173049 using real-time PCR technology. Expression levels of STRA6 and ISLR2 are significantly increased in specimens homozygous for the protective allele G compared to samples with A/G and A/A genotype. ( B ) Expression of STRA6 and ISLR2 mRNA in ocular tissues (iris and retina) derived from normal human donors (control, n = 42) and donors with PEX syndrome ( n = 24) using real-time PCR technology. Expression levels were significantly reduced in PEX specimens compared to control specimens. The relative expression levels were normalized relative to GAPDH and are represented as mean values ± SD ( * P < 0.05; ** P < 0.01; *** P < 0.001). ( C ) Expression of STRA6 protein in ocular tissues (retina, iris and ciliary body) of normal human donor eyes and donor eyes with PEX syndrome, as determined by immunofluorescence labeling. In normal eye tissues (control), STRA6 immunopositivity (green fluorescence) is observed in the RPE overlying the choroid (CH), retinal BVs (asterisk) within the RGC layer, iridal BVs (asterisk) in the iris ST and the inner layer of the CE adjacent to the ciliary ST. Expression levels are reduced in tissues of PEX eyes, and reduced staining intensities are often associated with LOXL1-positive PEX material accumulations (PEX, red immunofluorescence) in iris blood vessel walls and on the surface of the CE. ( D ) Expression of ISLR2 protein in ocular tissues (retina, iris and TM) of normal human donor eyes and donor eyes with PEX syndrome, as determined by immunofluorescence labeling. In normal control tissues, ISLR2 immunopositivity (green fluorescence) is observed in the retinal NFL, single cells (arrow) of the retinal ganglion cell layer (RGC) adjacent to BVs (asterisk), endothelial cells of BVs (asterisks) in the iris ST, and endothelial cells of the TM and SC. Markedly reduced staining intensities were seen in tissues of PEX eyes (INL means inner nuclear layer; ONL, outer nuclear layer; DAPI nuclear counterstain in blue).

Article Snippet: Cryosections of 4 μ m thickness were fixed in cold acetone for 10 min, washed in phosphate balanced saline (PBS) and permeabilized using 0.1% Triton X-100 in PBS for 10 min. After blocking with 10% normal goat serum, sections were incubated over night at 4°C in primary antibodies against STRA6 (ab169600, Abcam, Cambridge, UK), ISLR2 (ab81254, Abcam) and LOXL1 (kindly provided by Takako Sasaki, Department of Biochemistry II, Oita University, Japan) diluted in PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Immunofluorescence, Labeling, Fluorescence, Staining