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Expression of mSCARB2/hSCARB2 in <t>Scarb2-SCARB2</t> BAC Tg mice. (A) Schematic diagram showing the construction of the recombinant BAC. The RP23-228N3 clone is shown at the top. The genomic sequence spanning from exon 3 to exon 12 was replaced with the corresponding part of human SCARB2 cDNA that carries a FLAG tag-coding sequence. The resulting clone carrying the humanized Scarb2 transgene is displayed at the bottom. PCR genotyping of the Tg mice is also shown. Genomic DNAs isolated from Tg3 and non-Tg mice were subjected to PCR genotyping as described in Materials and Methods. The amplified product for the Tg sample is 1,511 bp long. NTC, no-template control. (B) Tissue distribution of the mSCARB2/hSCARB2 chimeric protein in Tg mice. (Top and middle) Tg and non-Tg mice were sacrificed, and cerebrum, cerebellum, spinal cord, heart, liver, spleen, lung, stomach, duodenum, jejunum, ileum, colon, kidney, and skeletal muscle were harvested for Western blotting with anti-hSCARB2 (top) and anti-FLAG (middle) antibodies. It should be noted that the anti-hSCARB2 antibody is known to cross-react with the murine endogenous protein. (Bottom) The immunoblot membrane shown in the top panel was stripped and reprobed with anti-SCARB2 antibody, which can react with both murine and human proteins. A representative result out of three experiments is shown. (C) IHC analysis of expression of the chimeric protein in Tg mice. Cerebral cortex, hippocampus, hypothalamus, medulla, thoracic spinal cord, stomach, duodenum, jejunum, ileum, colon, liver, spleen, skeletal muscle, thymus, and mesenteric lymph nodes were collected from the Tg mice; paraffinized; sectioned for immunohistochemical staining with the anti-hSCARB2 antibody; and counterstained with hematoxylin. Original magnification, ×200. A representative result out of three experiments is shown.
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Expression of mSCARB2/hSCARB2 in <t>Scarb2-SCARB2</t> BAC Tg mice. (A) Schematic diagram showing the construction of the recombinant BAC. The RP23-228N3 clone is shown at the top. The genomic sequence spanning from exon 3 to exon 12 was replaced with the corresponding part of human SCARB2 cDNA that carries a FLAG tag-coding sequence. The resulting clone carrying the humanized Scarb2 transgene is displayed at the bottom. PCR genotyping of the Tg mice is also shown. Genomic DNAs isolated from Tg3 and non-Tg mice were subjected to PCR genotyping as described in Materials and Methods. The amplified product for the Tg sample is 1,511 bp long. NTC, no-template control. (B) Tissue distribution of the mSCARB2/hSCARB2 chimeric protein in Tg mice. (Top and middle) Tg and non-Tg mice were sacrificed, and cerebrum, cerebellum, spinal cord, heart, liver, spleen, lung, stomach, duodenum, jejunum, ileum, colon, kidney, and skeletal muscle were harvested for Western blotting with anti-hSCARB2 (top) and anti-FLAG (middle) antibodies. It should be noted that the anti-hSCARB2 antibody is known to cross-react with the murine endogenous protein. (Bottom) The immunoblot membrane shown in the top panel was stripped and reprobed with anti-SCARB2 antibody, which can react with both murine and human proteins. A representative result out of three experiments is shown. (C) IHC analysis of expression of the chimeric protein in Tg mice. Cerebral cortex, hippocampus, hypothalamus, medulla, thoracic spinal cord, stomach, duodenum, jejunum, ileum, colon, liver, spleen, skeletal muscle, thymus, and mesenteric lymph nodes were collected from the Tg mice; paraffinized; sectioned for immunohistochemical staining with the anti-hSCARB2 antibody; and counterstained with hematoxylin. Original magnification, ×200. A representative result out of three experiments is shown.
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Expression of mSCARB2/hSCARB2 in <t>Scarb2-SCARB2</t> BAC Tg mice. (A) Schematic diagram showing the construction of the recombinant BAC. The RP23-228N3 clone is shown at the top. The genomic sequence spanning from exon 3 to exon 12 was replaced with the corresponding part of human SCARB2 cDNA that carries a FLAG tag-coding sequence. The resulting clone carrying the humanized Scarb2 transgene is displayed at the bottom. PCR genotyping of the Tg mice is also shown. Genomic DNAs isolated from Tg3 and non-Tg mice were subjected to PCR genotyping as described in Materials and Methods. The amplified product for the Tg sample is 1,511 bp long. NTC, no-template control. (B) Tissue distribution of the mSCARB2/hSCARB2 chimeric protein in Tg mice. (Top and middle) Tg and non-Tg mice were sacrificed, and cerebrum, cerebellum, spinal cord, heart, liver, spleen, lung, stomach, duodenum, jejunum, ileum, colon, kidney, and skeletal muscle were harvested for Western blotting with anti-hSCARB2 (top) and anti-FLAG (middle) antibodies. It should be noted that the anti-hSCARB2 antibody is known to cross-react with the murine endogenous protein. (Bottom) The immunoblot membrane shown in the top panel was stripped and reprobed with anti-SCARB2 antibody, which can react with both murine and human proteins. A representative result out of three experiments is shown. (C) IHC analysis of expression of the chimeric protein in Tg mice. Cerebral cortex, hippocampus, hypothalamus, medulla, thoracic spinal cord, stomach, duodenum, jejunum, ileum, colon, liver, spleen, skeletal muscle, thymus, and mesenteric lymph nodes were collected from the Tg mice; paraffinized; sectioned for immunohistochemical staining with the anti-hSCARB2 antibody; and counterstained with hematoxylin. Original magnification, ×200. A representative result out of three experiments is shown.
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Expression of mSCARB2/hSCARB2 in <t>Scarb2-SCARB2</t> BAC Tg mice. (A) Schematic diagram showing the construction of the recombinant BAC. The RP23-228N3 clone is shown at the top. The genomic sequence spanning from exon 3 to exon 12 was replaced with the corresponding part of human SCARB2 cDNA that carries a FLAG tag-coding sequence. The resulting clone carrying the humanized Scarb2 transgene is displayed at the bottom. PCR genotyping of the Tg mice is also shown. Genomic DNAs isolated from Tg3 and non-Tg mice were subjected to PCR genotyping as described in Materials and Methods. The amplified product for the Tg sample is 1,511 bp long. NTC, no-template control. (B) Tissue distribution of the mSCARB2/hSCARB2 chimeric protein in Tg mice. (Top and middle) Tg and non-Tg mice were sacrificed, and cerebrum, cerebellum, spinal cord, heart, liver, spleen, lung, stomach, duodenum, jejunum, ileum, colon, kidney, and skeletal muscle were harvested for Western blotting with anti-hSCARB2 (top) and anti-FLAG (middle) antibodies. It should be noted that the anti-hSCARB2 antibody is known to cross-react with the murine endogenous protein. (Bottom) The immunoblot membrane shown in the top panel was stripped and reprobed with anti-SCARB2 antibody, which can react with both murine and human proteins. A representative result out of three experiments is shown. (C) IHC analysis of expression of the chimeric protein in Tg mice. Cerebral cortex, hippocampus, hypothalamus, medulla, thoracic spinal cord, stomach, duodenum, jejunum, ileum, colon, liver, spleen, skeletal muscle, thymus, and mesenteric lymph nodes were collected from the Tg mice; paraffinized; sectioned for immunohistochemical staining with the anti-hSCARB2 antibody; and counterstained with hematoxylin. Original magnification, ×200. A representative result out of three experiments is shown.
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Expression of mSCARB2/hSCARB2 in Scarb2-SCARB2 BAC Tg mice. (A) Schematic diagram showing the construction of the recombinant BAC. The RP23-228N3 clone is shown at the top. The genomic sequence spanning from exon 3 to exon 12 was replaced with the corresponding part of human SCARB2 cDNA that carries a FLAG tag-coding sequence. The resulting clone carrying the humanized Scarb2 transgene is displayed at the bottom. PCR genotyping of the Tg mice is also shown. Genomic DNAs isolated from Tg3 and non-Tg mice were subjected to PCR genotyping as described in Materials and Methods. The amplified product for the Tg sample is 1,511 bp long. NTC, no-template control. (B) Tissue distribution of the mSCARB2/hSCARB2 chimeric protein in Tg mice. (Top and middle) Tg and non-Tg mice were sacrificed, and cerebrum, cerebellum, spinal cord, heart, liver, spleen, lung, stomach, duodenum, jejunum, ileum, colon, kidney, and skeletal muscle were harvested for Western blotting with anti-hSCARB2 (top) and anti-FLAG (middle) antibodies. It should be noted that the anti-hSCARB2 antibody is known to cross-react with the murine endogenous protein. (Bottom) The immunoblot membrane shown in the top panel was stripped and reprobed with anti-SCARB2 antibody, which can react with both murine and human proteins. A representative result out of three experiments is shown. (C) IHC analysis of expression of the chimeric protein in Tg mice. Cerebral cortex, hippocampus, hypothalamus, medulla, thoracic spinal cord, stomach, duodenum, jejunum, ileum, colon, liver, spleen, skeletal muscle, thymus, and mesenteric lymph nodes were collected from the Tg mice; paraffinized; sectioned for immunohistochemical staining with the anti-hSCARB2 antibody; and counterstained with hematoxylin. Original magnification, ×200. A representative result out of three experiments is shown.

Journal: Journal of Virology

Article Title: A Novel Murine Model Expressing a Chimeric mSCARB2/hSCARB2 Receptor Is Highly Susceptible to Oral Infection with Clinical Isolates of Enterovirus 71

doi: 10.1128/JVI.00183-19

Figure Lengend Snippet: Expression of mSCARB2/hSCARB2 in Scarb2-SCARB2 BAC Tg mice. (A) Schematic diagram showing the construction of the recombinant BAC. The RP23-228N3 clone is shown at the top. The genomic sequence spanning from exon 3 to exon 12 was replaced with the corresponding part of human SCARB2 cDNA that carries a FLAG tag-coding sequence. The resulting clone carrying the humanized Scarb2 transgene is displayed at the bottom. PCR genotyping of the Tg mice is also shown. Genomic DNAs isolated from Tg3 and non-Tg mice were subjected to PCR genotyping as described in Materials and Methods. The amplified product for the Tg sample is 1,511 bp long. NTC, no-template control. (B) Tissue distribution of the mSCARB2/hSCARB2 chimeric protein in Tg mice. (Top and middle) Tg and non-Tg mice were sacrificed, and cerebrum, cerebellum, spinal cord, heart, liver, spleen, lung, stomach, duodenum, jejunum, ileum, colon, kidney, and skeletal muscle were harvested for Western blotting with anti-hSCARB2 (top) and anti-FLAG (middle) antibodies. It should be noted that the anti-hSCARB2 antibody is known to cross-react with the murine endogenous protein. (Bottom) The immunoblot membrane shown in the top panel was stripped and reprobed with anti-SCARB2 antibody, which can react with both murine and human proteins. A representative result out of three experiments is shown. (C) IHC analysis of expression of the chimeric protein in Tg mice. Cerebral cortex, hippocampus, hypothalamus, medulla, thoracic spinal cord, stomach, duodenum, jejunum, ileum, colon, liver, spleen, skeletal muscle, thymus, and mesenteric lymph nodes were collected from the Tg mice; paraffinized; sectioned for immunohistochemical staining with the anti-hSCARB2 antibody; and counterstained with hematoxylin. Original magnification, ×200. A representative result out of three experiments is shown.

Article Snippet: For detection of mSCARB2, a goat anti-SCARB2 antibody (catalog no. sc-25867; Santa Cruz Biotechnology, TX, USA) was used.

Techniques: Expressing, Recombinant, Sequencing, FLAG-tag, Isolation, Amplification, Control, Western Blot, Membrane, Immunohistochemical staining, Staining