Human LIMPII/SR-B2 Antibody

Detection of LIMPII/SR-B2 by Western Blot Generation of a hybrid mouse model SCARB2/stat-1 KO by crossbreeding hSCARB2 transgenic mice and stat-1 KO mice.(a) Human SCARB2 (hSCARB2) cDNA was cloned under its own native promoter in an SV40 expression vector. (b) The generation of homozygous hSCARB2+/+ transgenic mice was as detailed in M&M. The heterozygote strain of hSCARB2+/−/stat-1+/− mice were generated by crossing the stat-1−/− and the hSCARB2+/+ parental mice. The hybrid strain hSCARB2+/+/stat-1−/− was generated by crossing the heterozygote mice hSCARB2+/−/stat-1+/− to each other. (c) Genotyping of parental and hybrid mouse strains was performed by PCR assay using genomic DNAs extracted from mouse tail. The transgene of hSCARB2 was screened by detection of a 369 bp PCR product using primers specific for hSCARB2. For the stat-1+/− heterozygote mice, 320 bp and 150 bp PCR products were used as markers for screening. The former indicates a mutant stat-1 allele, while the latter indicates a wild type stat-1 allele. (d) The expressions of hSCARB2 protein were compared among parental and hybrid mouse strains in brain, spinal cord, spleen and muscle by immunoblot via an anti-SCARB2 antibody. The weaker signals of the SCARB2 protein detected in stat-1 KO mice reflect cross reactivity between human and mouse SCARB2 proteins to the anti-SCARB2 antibody. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27499235), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of LIMPII/SR-B2 by Western Blot Generation of a hybrid mouse model SCARB2/stat-1 KO by crossbreeding hSCARB2 transgenic mice and stat-1 KO mice.(a) Human SCARB2 (hSCARB2) cDNA was cloned under its own native promoter in an SV40 expression vector. (b) The generation of homozygous hSCARB2+/+ transgenic mice was as detailed in M&M. The heterozygote strain of hSCARB2+/−/stat-1+/− mice were generated by crossing the stat-1−/− and the hSCARB2+/+ parental mice. The hybrid strain hSCARB2+/+/stat-1−/− was generated by crossing the heterozygote mice hSCARB2+/−/stat-1+/− to each other. (c) Genotyping of parental and hybrid mouse strains was performed by PCR assay using genomic DNAs extracted from mouse tail. The transgene of hSCARB2 was screened by detection of a 369 bp PCR product using primers specific for hSCARB2. For the stat-1+/− heterozygote mice, 320 bp and 150 bp PCR products were used as markers for screening. The former indicates a mutant stat-1 allele, while the latter indicates a wild type stat-1 allele. (d) The expressions of hSCARB2 protein were compared among parental and hybrid mouse strains in brain, spinal cord, spleen and muscle by immunoblot via an anti-SCARB2 antibody. The weaker signals of the SCARB2 protein detected in stat-1 KO mice reflect cross reactivity between human and mouse SCARB2 proteins to the anti-SCARB2 antibody. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27499235), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of LIMPII/SR-B2 by Western Blot HS and SCARB2 expression in genetically modified RD-A cells.(A–B) FACS analysis of HS expression at the cell surface. (C) Western blotting analysis using anti-SCARB2 (top), anti-flag (middle), and anti-beta -actin (bottom) antibodies. The arrowheads indicate hSCARB2. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32187235), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of LIMPII/SR-B2 by Western Blot The reduction of endogenous hSCARB2 and KRM1 expression impacted CVA10 infection. RD Cells were transfected individually with 50 pmoles of siRNA specific to hSCARB2 (s2651(S1) and s2652(S2)), KRM1 (s38393(K1) and s38394(K2)), or mixed specific siRNA with equal amounts (50 + 50 pmoles) of S1 + K1, S1 + K2, S2 + K1, S2 + K2, or negative siRNA, followed by 48 h of incubation. Infection of siRNA-treated cells with CVA10 (MOI = 0.05) and then cultured for another 24 h then proceeded. (a) Western blotting with the respective antibody examined the expression level of hSCARB2 or KRN1 in the cells. (b) The supernatant and (c) lysate were collected and subjected to a plaque-forming assay to detect the amounts of produced CVA10, and the results were shown. (d) Total amounts of CVA10 production using the sum of the viral amounts from (b,c) are shown. The symbols **, *** and **** were used to indicate p < 0.01, p < 0.001, and p < 0.0001, respectively. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37112912), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of LIMPII/SR-B2 by Western Blot Cross-linking mass spectrometry analysis of lysosome-enriched fractions. A Experimental workflow for the XL-LC-MS/MS analysis of lysosome-enriched fractions. Created with BioRender.com. b Normalized beta -hexosaminidase activities for individual fractions from lysosome enrichment by SPIONs. Data are presented as mean values + SD (n = 3, biologically independent samples over three independent experiments). c Western blot analysis of lysosome-enriched fractions for contamination by other organelles (n = 2). Lysosome: lysosomal proteins (CTSD, LAMP2, LIMP2, and LAMTOR1). Other: Golgi apparatus (GM130), cytoskeleton (TUBA), cytosol (GAPDH), endoplasmic reticulum (CANX), and mitochondria (SDHA). d Summed iBAQ abundances for proteins identified in lysosome-enriched fractions in ≥3 replicates. e Classification of unique cross-linked residue pairs. f Proteins detected in non-cross-linked lysosome-enriched fractions (proteome), and unique lysosomal cross-linked residue pairs (interactome) for DR and IT samples. g Localization of CSMs for 68 lysosomal proteins cross-linked in the DR and IT state. Cytosolic: proteins located at the cytosolic face of the lysosomal membrane; Lumen: lysosomal luminal proteins. h Correlation of cross-link identification and protein abundance for lysosomal proteins. CSMs and PSMs represent summed values of the analysis (n = 6, biologically independent samples over six independent experiments (3× DR and 3× IT)). SPIONs superparamagnetic iron oxide nanoparticles, DR disrupted, IT intact, SCX strong cation-exchange, IN input, FT flow through, W wash, EL eluate, WCL whole-cell lysate, iBAQ intensity-based absolute quantification, XLs cross-links, CSMs cross-link spectral matches, PSMs peptide spectral matches. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36266287), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of LIMPII/SR-B2 by Western Blot The reduction of endogenous hSCARB2 and KRM1 expression impacted CVA10 infection. RD Cells were transfected individually with 50 pmoles of siRNA specific to hSCARB2 (s2651(S1) and s2652(S2)), KRM1 (s38393(K1) and s38394(K2)), or mixed specific siRNA with equal amounts (50 + 50 pmoles) of S1 + K1, S1 + K2, S2 + K1, S2 + K2, or negative siRNA, followed by 48 h of incubation. Infection of siRNA-treated cells with CVA10 (MOI = 0.05) and then cultured for another 24 h then proceeded. (a) Western blotting with the respective antibody examined the expression level of hSCARB2 or KRN1 in the cells. (b) The supernatant and (c) lysate were collected and subjected to a plaque-forming assay to detect the amounts of produced CVA10, and the results were shown. (d) Total amounts of CVA10 production using the sum of the viral amounts from (b,c) are shown. The symbols **, *** and **** were used to indicate p < 0.01, p < 0.001, and p < 0.0001, respectively. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37112912), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of LIMPII/SR-B2 by Western Blot Cross-linking mass spectrometry analysis of lysosome-enriched fractions. A Experimental workflow for the XL-LC-MS/MS analysis of lysosome-enriched fractions. Created with BioRender.com. b Normalized beta -hexosaminidase activities for individual fractions from lysosome enrichment by SPIONs. Data are presented as mean values + SD (n = 3, biologically independent samples over three independent experiments). c Western blot analysis of lysosome-enriched fractions for contamination by other organelles (n = 2). Lysosome: lysosomal proteins (CTSD, LAMP2, LIMP2, and LAMTOR1). Other: Golgi apparatus (GM130), cytoskeleton (TUBA), cytosol (GAPDH), endoplasmic reticulum (CANX), and mitochondria (SDHA). d Summed iBAQ abundances for proteins identified in lysosome-enriched fractions in ≥3 replicates. e Classification of unique cross-linked residue pairs. f Proteins detected in non-cross-linked lysosome-enriched fractions (proteome), and unique lysosomal cross-linked residue pairs (interactome) for DR and IT samples. g Localization of CSMs for 68 lysosomal proteins cross-linked in the DR and IT state. Cytosolic: proteins located at the cytosolic face of the lysosomal membrane; Lumen: lysosomal luminal proteins. h Correlation of cross-link identification and protein abundance for lysosomal proteins. CSMs and PSMs represent summed values of the analysis (n = 6, biologically independent samples over six independent experiments (3× DR and 3× IT)). SPIONs superparamagnetic iron oxide nanoparticles, DR disrupted, IT intact, SCX strong cation-exchange, IN input, FT flow through, W wash, EL eluate, WCL whole-cell lysate, iBAQ intensity-based absolute quantification, XLs cross-links, CSMs cross-link spectral matches, PSMs peptide spectral matches. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36266287), licensed under a CC-BY license. Not internally tested by R&D Systems.