Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Schematic of COOH-terminal V5-His-tagged hCR-1 (CR1-V5), CR-CFC (CR-CFC-V5) and a soluble form of hCR-1 (CR1ΔC). Sequences from hCR-1 are shown in red and from hCFC1 in blue. (B–C) Western blot analysis of cell lysates (B) and conditioned medium (C) from 293T cells which had been transiently transfected with indicated constructs. Immunoblot was performed with anti-hCR-1 mAb (upper panel) and the same blots were then stripped and reblotted with an anti-V5 mAb (lower panel). (D) Triton-X114 phase separation analysis of cell lysates from 293T cells which had been transfected with indicated constructs. Triton-X114 phase separation was carried out as described in Materials and Methods, and aqueous phase (A) and detergent phase (D) were analyzed by Western blotting. Immunoblot was performed with the anti-hCR-1 mAb (upper panel) and the same blots were then stripped and reblotted with the anti-V5 mAb (middle panel). Ponceau S staining of the same blot is shown in the bottom. (D) Effect of PI-PLC on release of CR-CFC and CR-CFC-V5. 293T cells were transiently transfected with the indicated constructs and PI-PLC treatment (1 unit/mL) was performed. Cells and supernatants were collected and analyzed by Western blotting with an anti-CR-1 specific mAb.

Article Snippet: After blocking with 5% milk, hCR-1-related proteins were detected with an anti-hCR-1 mouse monoclonal antibody (mAb) (B3F6, Biogen Idec., Cambridge, MA) at a 1:5000 dilution or with an anti-V5 mouse mAb (Invitrogen) at a 1:5000 dilution and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Amersham Biosciences, Piscataway, NJ). hCFC1-realted proteins were detected with a sheep anti-hCFC1 polyclonal antibody (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL and anti-sheep IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Santa Cruz, Santa Cruz, CA).

Techniques: Western Blot, Transfection, Construct, Staining

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A–B) Comparison of the cell-surface expression of CR1WT and CR1-V5 (A) or CR-CFC and CR-CFC-V5 (B) in transiently transfected 293T cells. (C–D) 293T cells which had been transiently transfected with CR1-V5 (C) or CR-CFC-V5 (D) were collected with enzyme-free cell dissociation buffer and incubated in PBS with or without 1 unit/mL PI-PLC at 37°C for 30 min. After centrifugation, cells were collected and stained with PE-conjugated anti-hCR-1 mAb followed by FACS analysis. Empty vector (EV) was used for a negative control.

Article Snippet: After blocking with 5% milk, hCR-1-related proteins were detected with an anti-hCR-1 mouse monoclonal antibody (mAb) (B3F6, Biogen Idec., Cambridge, MA) at a 1:5000 dilution or with an anti-V5 mouse mAb (Invitrogen) at a 1:5000 dilution and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Amersham Biosciences, Piscataway, NJ). hCFC1-realted proteins were detected with a sheep anti-hCFC1 polyclonal antibody (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL and anti-sheep IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Santa Cruz, Santa Cruz, CA).

Techniques: Expressing, Transfection, Incubation, Centrifugation, Staining, Plasmid Preparation, Negative Control

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A–B) Western blot analysis of cell lysates (A) and conditioned medium (B) from transiently transfected 293T cells. Immunoblot was performed with anti-FLAG mAb (upper panel) and the same blots were then stripped and reblotted with an anti-V5 mAb (lower panel). (D) [3H]-ethanolamine Metabolic labeling. 293T cells were transiently transfected with indicated constructs and labeled with [3H]-ethanolamine (100 μCi) for 24 h. The cell lysates were subjected to immunoprecipitation using anti-Flag M2-conjugated agarose. After dissolving on SDS-PAGE, the samples were analyzed by autoradiography. *; non-specific bands.

Article Snippet: After blocking with 5% milk, hCR-1-related proteins were detected with an anti-hCR-1 mouse monoclonal antibody (mAb) (B3F6, Biogen Idec., Cambridge, MA) at a 1:5000 dilution or with an anti-V5 mouse mAb (Invitrogen) at a 1:5000 dilution and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Amersham Biosciences, Piscataway, NJ). hCFC1-realted proteins were detected with a sheep anti-hCFC1 polyclonal antibody (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL and anti-sheep IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Santa Cruz, Santa Cruz, CA).

Techniques: Western Blot, Transfection, Labeling, Construct, Immunoprecipitation, SDS Page, Autoradiography

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A–H) Immunocytochemistry of indicated hCR-1 variants in transiently transfected 293T cells was performed after permeabilization by Triton X-100. CR-1 proteins were stained with anti-CR-1 mAb (A1–D1, green) or anti-V5 antibody (E1–F1, green). DsRed-Golgi was co-transfected in A2–F2 (red). Co-staining of V5-tagged constructs with anti-CR-1 mAb (G1–H1, green) and anti-V5 antibody (G2–H2, red) is shown in G and H. Nuclei were counterstained with DAPI (blue) and tricolor merged images are shown in A3–H3. Images were visualized by confocal microscopy. Arrowheads indicate Golgi localization of CR-1 variant protein. Scale bar = 10 μm. (I–J) Immunocytochemistry of hCFC1 and CFC1-V5 in transiently transfected 293T cells was performed using anti-human CFC1 specific antibody and anti-V5 mAb. DsRed-Golgi was co-transfected in I2 (red). Arrowheads indicate Golgi localization of CFC1 or CFC1-V5 proteins.

Article Snippet: After blocking with 5% milk, hCR-1-related proteins were detected with an anti-hCR-1 mouse monoclonal antibody (mAb) (B3F6, Biogen Idec., Cambridge, MA) at a 1:5000 dilution or with an anti-V5 mouse mAb (Invitrogen) at a 1:5000 dilution and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Amersham Biosciences, Piscataway, NJ). hCFC1-realted proteins were detected with a sheep anti-hCFC1 polyclonal antibody (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL and anti-sheep IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Santa Cruz, Santa Cruz, CA).

Techniques: Immunocytochemistry, Transfection, Staining, Construct, Confocal Microscopy, Variant Assay

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Schematic of COOH-terminally deleted CR-CFC or CFC1 construct. Sequences from hCR-1 are shown in red and from hCFC1 in blue. (B) Expression of each CR-CFC variant in cell lysates and conditioned medium. 293T cells were transiently transfected with indicated expression vectors. Total cell lysates (CL) and conditioned medium (CM) were collected 24 h after transfection. Immunoblot analysis was performed with the anti-hCR-1 antibody (upper panel) and the same blots were then stripped and reblotted with the anti-V5 antibody (lower panel). (C) FACS analysis of cell-surface expression for each CR-CFC variant. 293T cells transiently transfected with the indicated expression vectors were stained with PE-conjugated anti-hCR-1 mAb and analyzed with FACS. EV was used as a negative control. (D–F) Immunocytochemical analysis of each CR-CFC variant in transiently transfected 293T cells after permeabilization. CR-1 proteins were stained with anti-CR-1 mAb (D1–E1) (green). DsRed-Golgi was co-transfected with each CR-CFC construct in D2–E2 (red). Co-staining of CR-CFC L191-V5 with anti-CR-1 mAb (F1, green) and anti-V5 antibody (F2, red) is shown in F. Nuclei were counterstained with DAPI (blue) and tricolor merged images are shown in D3–F3. Images were visualized by confocal microscopy. Arrowheads indicate Golgi localization of the CR-CFC protein. Scale bar = 10 μm. (G) (n2)7-Luc reporter assay. Serum starved 293T cells were transiently transfected with the (n2)7-Luc reporter, TK renilla reporter, mFast-1, mNodal-V5, ALK4-HA expression vector and the indicated expression vectors. Values indicate fold induction of relative luciferase unit against EV. Mean ± SD was shown for three independent experiments. P values were calculated with Student’s T-test.

Article Snippet: After blocking with 5% milk, hCR-1-related proteins were detected with an anti-hCR-1 mouse monoclonal antibody (mAb) (B3F6, Biogen Idec., Cambridge, MA) at a 1:5000 dilution or with an anti-V5 mouse mAb (Invitrogen) at a 1:5000 dilution and anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Amersham Biosciences, Piscataway, NJ). hCFC1-realted proteins were detected with a sheep anti-hCFC1 polyclonal antibody (R&D Systems, Minneapolis, MN) at a concentration of 0.2 μg/mL and anti-sheep IgG horseradish peroxidase-conjugated secondary antibody (1:3000, Santa Cruz, Santa Cruz, CA).

Techniques: Construct, Expressing, Variant Assay, Transfection, Western Blot, Staining, Negative Control, Confocal Microscopy, Reporter Assay, Plasmid Preparation, Luciferase

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Effect of PI-PLC on release of wild-type hCFC1. 293T cells transiently transfected with the hCFC1 expression vector were collected with enzyme-free cell dissociation buffer and incubated in PBS with the indicated concentration of PI-PLC at 37°C for 30 min. After centrifugation, cells and supernatants were collected and Western blot analysis was carried out using an anti-human CFC1 polyclonal antibody. (B) FACS analysis of cell-surface expression of hCFC1 in transiently transfected 293T cells with or without PI-PLC treatment. 293T cells transfected with the indicated constructs were collected and treated with or without PI-PLC (1 unit/mL). After centrifugation, cells were stained with an anti-human CFC1 polyclonal antibody and Alexa Fluor 488-conjugated secondary antibody, and FACS analysis was performed. Arrows indicate the two peaks of the high and low expression population. (C) [3H]-ethanolamine Metabolic labeling. 293T cells were transiently transfected with indicated constructs and labeled with [3H]-ethanolamine (100 μCi) for 24 h. The cell lysates were subjected to immunoprecipitation using anti-FLAG M2-conjugated agarose. After dissolving on SDS-PAGE, the samples were analyzed by autoradiography. *; non-specific bands.

Article Snippet: For co-staining with anti-hCR-1 mAb (IgG 1 ) and anti-V5 mAb (IgG 2a ), samples were first stained with anti-V5 mAb and Alexa Fluor 596-conjugated anti-mouse whole IgG secondary antibody (1:200, Invitrogen) and then stained with anti-hCR-1 mAb and Alexa Fluor 488-conjugated anti-mouse IgG 1 -specific secondary antibody (1:200, Invitrogen). hCFC1 protein was stained with 25 μg/mL of the sheep anti-hCFC1 polyclonal antibody (R&D Systems) and anti-sheep IgG Alexa Fluor 488 conjugate (1:200, Invitrogen).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Concentration Assay, Centrifugation, Western Blot, Construct, Staining, Labeling, Immunoprecipitation, SDS Page, Autoradiography

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Effect of PI-PLC on release of wild-type hCFC1. 293T cells transiently transfected with the hCFC1 expression vector were collected with enzyme-free cell dissociation buffer and incubated in PBS with the indicated concentration of PI-PLC at 37°C for 30 min. After centrifugation, cells and supernatants were collected and Western blot analysis was carried out using an anti-human CFC1 polyclonal antibody. (B) FACS analysis of cell-surface expression of hCFC1 in transiently transfected 293T cells with or without PI-PLC treatment. 293T cells transfected with the indicated constructs were collected and treated with or without PI-PLC (1 unit/mL). After centrifugation, cells were stained with an anti-human CFC1 polyclonal antibody and Alexa Fluor 488-conjugated secondary antibody, and FACS analysis was performed. Arrows indicate the two peaks of the high and low expression population. (C) [3H]-ethanolamine Metabolic labeling. 293T cells were transiently transfected with indicated constructs and labeled with [3H]-ethanolamine (100 μCi) for 24 h. The cell lysates were subjected to immunoprecipitation using anti-FLAG M2-conjugated agarose. After dissolving on SDS-PAGE, the samples were analyzed by autoradiography. *; non-specific bands.

Article Snippet: For co-staining with anti-hCR-1 mAb (IgG 1 ) and anti-V5 mAb (IgG 2a ), samples were first stained with anti-V5 mAb and Alexa Fluor 596-conjugated anti-mouse whole IgG secondary antibody (1:200, Invitrogen) and then stained with anti-hCR-1 mAb and Alexa Fluor 488-conjugated anti-mouse IgG 1 -specific secondary antibody (1:200, Invitrogen). hCFC1 protein was stained with 25 μg/mL of the sheep anti-hCFC1 polyclonal antibody (R&D Systems) and anti-sheep IgG Alexa Fluor 488 conjugate (1:200, Invitrogen).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Concentration Assay, Centrifugation, Western Blot, Construct, Staining, Labeling, Immunoprecipitation, SDS Page, Autoradiography

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Schematic of hCR-1 (CR1WT), hCFC1 (CFC1WT), a chimeric construct (CR-CFC), and a transmembrane form of hCR-1 (CR1TM). Sequences from hCR-1 are shown in red and from hCFC1 in blue. (B) Effect of PI-PLC on release of CR1WT and CR-CFC. 293T cells were transiently transfected with indicated constructs and PI-PLC treatment (1 unit/mL) was performed. Cells and supernatants were collected and analyzed by Western blotting with an anti-CR-1 specific mAb. (C) FACS analysis of cell-surface expression of CR-1 variants in transiently transfected 293T cells with or without PI-PLC treatment. 293T cells transfected with the indicated constructs were collected and treated with or without PI-PLC (1 unit/mL). Cells were stained with PE-conjugated anti-hCR-1 mAb followed by FACS analysis. Arrows indicate the two peaks of the high and low expression population in CR1WT, CR-CFC and CR1TM, respectively.

Article Snippet: For co-staining with anti-hCR-1 mAb (IgG 1 ) and anti-V5 mAb (IgG 2a ), samples were first stained with anti-V5 mAb and Alexa Fluor 596-conjugated anti-mouse whole IgG secondary antibody (1:200, Invitrogen) and then stained with anti-hCR-1 mAb and Alexa Fluor 488-conjugated anti-mouse IgG 1 -specific secondary antibody (1:200, Invitrogen). hCFC1 protein was stained with 25 μg/mL of the sheep anti-hCFC1 polyclonal antibody (R&D Systems) and anti-sheep IgG Alexa Fluor 488 conjugate (1:200, Invitrogen).

Techniques: Construct, Transfection, Western Blot, Expressing, Staining

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Schematic of COOH-terminal V5-His-tagged hCR-1 (CR1-V5), CR-CFC (CR-CFC-V5) and a soluble form of hCR-1 (CR1ΔC). Sequences from hCR-1 are shown in red and from hCFC1 in blue. (B–C) Western blot analysis of cell lysates (B) and conditioned medium (C) from 293T cells which had been transiently transfected with indicated constructs. Immunoblot was performed with anti-hCR-1 mAb (upper panel) and the same blots were then stripped and reblotted with an anti-V5 mAb (lower panel). (D) Triton-X114 phase separation analysis of cell lysates from 293T cells which had been transfected with indicated constructs. Triton-X114 phase separation was carried out as described in Materials and Methods, and aqueous phase (A) and detergent phase (D) were analyzed by Western blotting. Immunoblot was performed with the anti-hCR-1 mAb (upper panel) and the same blots were then stripped and reblotted with the anti-V5 mAb (middle panel). Ponceau S staining of the same blot is shown in the bottom. (D) Effect of PI-PLC on release of CR-CFC and CR-CFC-V5. 293T cells were transiently transfected with the indicated constructs and PI-PLC treatment (1 unit/mL) was performed. Cells and supernatants were collected and analyzed by Western blotting with an anti-CR-1 specific mAb.

Article Snippet: For co-staining with anti-hCR-1 mAb (IgG 1 ) and anti-V5 mAb (IgG 2a ), samples were first stained with anti-V5 mAb and Alexa Fluor 596-conjugated anti-mouse whole IgG secondary antibody (1:200, Invitrogen) and then stained with anti-hCR-1 mAb and Alexa Fluor 488-conjugated anti-mouse IgG 1 -specific secondary antibody (1:200, Invitrogen). hCFC1 protein was stained with 25 μg/mL of the sheep anti-hCFC1 polyclonal antibody (R&D Systems) and anti-sheep IgG Alexa Fluor 488 conjugate (1:200, Invitrogen).

Techniques: Western Blot, Transfection, Construct, Staining

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A–H) Immunocytochemistry of indicated hCR-1 variants in transiently transfected 293T cells was performed after permeabilization by Triton X-100. CR-1 proteins were stained with anti-CR-1 mAb (A1–D1, green) or anti-V5 antibody (E1–F1, green). DsRed-Golgi was co-transfected in A2–F2 (red). Co-staining of V5-tagged constructs with anti-CR-1 mAb (G1–H1, green) and anti-V5 antibody (G2–H2, red) is shown in G and H. Nuclei were counterstained with DAPI (blue) and tricolor merged images are shown in A3–H3. Images were visualized by confocal microscopy. Arrowheads indicate Golgi localization of CR-1 variant protein. Scale bar = 10 μm. (I–J) Immunocytochemistry of hCFC1 and CFC1-V5 in transiently transfected 293T cells was performed using anti-human CFC1 specific antibody and anti-V5 mAb. DsRed-Golgi was co-transfected in I2 (red). Arrowheads indicate Golgi localization of CFC1 or CFC1-V5 proteins.

Article Snippet: For co-staining with anti-hCR-1 mAb (IgG 1 ) and anti-V5 mAb (IgG 2a ), samples were first stained with anti-V5 mAb and Alexa Fluor 596-conjugated anti-mouse whole IgG secondary antibody (1:200, Invitrogen) and then stained with anti-hCR-1 mAb and Alexa Fluor 488-conjugated anti-mouse IgG 1 -specific secondary antibody (1:200, Invitrogen). hCFC1 protein was stained with 25 μg/mL of the sheep anti-hCFC1 polyclonal antibody (R&D Systems) and anti-sheep IgG Alexa Fluor 488 conjugate (1:200, Invitrogen).

Techniques: Immunocytochemistry, Transfection, Staining, Construct, Confocal Microscopy, Variant Assay

Journal: Biochimica et Biophysica Acta

Article Title: Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

doi: 10.1016/j.bbamem.2008.09.011

Figure Lengend Snippet: (A) Schematic of COOH-terminally deleted CR-CFC or CFC1 construct. Sequences from hCR-1 are shown in red and from hCFC1 in blue. (B) Expression of each CR-CFC variant in cell lysates and conditioned medium. 293T cells were transiently transfected with indicated expression vectors. Total cell lysates (CL) and conditioned medium (CM) were collected 24 h after transfection. Immunoblot analysis was performed with the anti-hCR-1 antibody (upper panel) and the same blots were then stripped and reblotted with the anti-V5 antibody (lower panel). (C) FACS analysis of cell-surface expression for each CR-CFC variant. 293T cells transiently transfected with the indicated expression vectors were stained with PE-conjugated anti-hCR-1 mAb and analyzed with FACS. EV was used as a negative control. (D–F) Immunocytochemical analysis of each CR-CFC variant in transiently transfected 293T cells after permeabilization. CR-1 proteins were stained with anti-CR-1 mAb (D1–E1) (green). DsRed-Golgi was co-transfected with each CR-CFC construct in D2–E2 (red). Co-staining of CR-CFC L191-V5 with anti-CR-1 mAb (F1, green) and anti-V5 antibody (F2, red) is shown in F. Nuclei were counterstained with DAPI (blue) and tricolor merged images are shown in D3–F3. Images were visualized by confocal microscopy. Arrowheads indicate Golgi localization of the CR-CFC protein. Scale bar = 10 μm. (G) (n2)7-Luc reporter assay. Serum starved 293T cells were transiently transfected with the (n2)7-Luc reporter, TK renilla reporter, mFast-1, mNodal-V5, ALK4-HA expression vector and the indicated expression vectors. Values indicate fold induction of relative luciferase unit against EV. Mean ± SD was shown for three independent experiments. P values were calculated with Student’s T-test.

Article Snippet: For co-staining with anti-hCR-1 mAb (IgG 1 ) and anti-V5 mAb (IgG 2a ), samples were first stained with anti-V5 mAb and Alexa Fluor 596-conjugated anti-mouse whole IgG secondary antibody (1:200, Invitrogen) and then stained with anti-hCR-1 mAb and Alexa Fluor 488-conjugated anti-mouse IgG 1 -specific secondary antibody (1:200, Invitrogen). hCFC1 protein was stained with 25 μg/mL of the sheep anti-hCFC1 polyclonal antibody (R&D Systems) and anti-sheep IgG Alexa Fluor 488 conjugate (1:200, Invitrogen).

Techniques: Construct, Expressing, Variant Assay, Transfection, Western Blot, Staining, Negative Control, Confocal Microscopy, Reporter Assay, Plasmid Preparation, Luciferase