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igg2a against human cd59  (Bio-Rad)


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    Bio-Rad igg2a against human cd59
    Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein <t>CD59</t> in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).
    Igg2a Against Human Cd59, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg2a against human cd59/product/Bio-Rad
    Average 96 stars, based on 845 article reviews
    igg2a against human cd59 - by Bioz Stars, 2026-03
    96/100 stars

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    1) Product Images from "Selective export of human GPI-anchored proteins from the endoplasmic reticulum."

    Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

    Journal: Journal of cell science

    doi: 10.1242/jcs.062950

    Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein CD59 in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).
    Figure Legend Snippet: Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein CD59 in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).

    Techniques Used: Transfection, Immunoprecipitation, SDS Page, Autoradiography, Pulse Chase, Control

    Fig. 3. CD59 interacts with p24 and p23. HeLa cells were either untransfected (–) or transfected (+) with Myc-p23 (A,B,E), Myc- p23 and CD59-GFP (C), HA-p24 (D,F), HA-p24 and CD59-GFP (D), as indicated. After 48 hours the cells were subjected to immunoprecipitation (Ip), SDS-PAGE, and western blotting (Wb) using the indicated antibodies. (E,F)Cells were pulsed with [35S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc- p23 and HA-p24 pulled down newly synthesized endogenous CD59 and vice versa. *Bands that probably correspond to endogenous p24 and p23. IgGH, heavy chain of immunoglobulins; IgGL, light chain of immunoglobulins. Positions of molecular mass markers are indicated on the left.
    Figure Legend Snippet: Fig. 3. CD59 interacts with p24 and p23. HeLa cells were either untransfected (–) or transfected (+) with Myc-p23 (A,B,E), Myc- p23 and CD59-GFP (C), HA-p24 (D,F), HA-p24 and CD59-GFP (D), as indicated. After 48 hours the cells were subjected to immunoprecipitation (Ip), SDS-PAGE, and western blotting (Wb) using the indicated antibodies. (E,F)Cells were pulsed with [35S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc- p23 and HA-p24 pulled down newly synthesized endogenous CD59 and vice versa. *Bands that probably correspond to endogenous p24 and p23. IgGH, heavy chain of immunoglobulins; IgGL, light chain of immunoglobulins. Positions of molecular mass markers are indicated on the left.

    Techniques Used: Transfection, Immunoprecipitation, SDS Page, Western Blot, Autoradiography, Synthesized

    Fig. 6. Co-partitioning of GPI-anchored proteins and p24-p23 into lipid rafts. HeLa cells were untransfected (A) or were transfected with Myc-p23 (B) or with HA-p24 (C). Cells were lysed in MBS-Triton X-100 at 4°C and analyzed by sucrose gradient centrifugation (see Materials and Methods). Fractions were collected and separated by SDS-PAGE followed by western blotting with antibodies against the indicated proteins. In parallel, the lipid raft fraction (15%+20%+25% sucrose) was collected, immunoprecipitated with anti-CD59 antibody (B and C), separated by SDS-PAGE, and immunoblotted with anti-CD59 and anti-Myc antibodies (B) or with anti-CD59 and HA antibodies (C). IgGL: light chain of immunglobulins. (D)Untransfected cells [to detect endogenous CD59, transferrin receptor (TfR), ERGIC-53, and BAP- 31] or cells transfected with Myc-p23 or HA-p24 were pulsed for 10 minutes with [35S]methionine-cysteine and subjected to sucrose density gradient centrifugation (as in A). Fractions were immunoprecipitated with antibodies against the indicated proteins or Myc and HA, and separated by SDS-PAGE followed by autoradiography. 40+endo-H, immunoprecipitated CD59 and TfR were digested with endo-H (+endo-H) before analysis. (E)Lipid raft fractions (15+20+25) from 15-minute pulse-labeled cells were pooled, immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. Note co-immunoprecipitation with CD59. Positions of molecular mass markers (in kDa) are shown on the left.
    Figure Legend Snippet: Fig. 6. Co-partitioning of GPI-anchored proteins and p24-p23 into lipid rafts. HeLa cells were untransfected (A) or were transfected with Myc-p23 (B) or with HA-p24 (C). Cells were lysed in MBS-Triton X-100 at 4°C and analyzed by sucrose gradient centrifugation (see Materials and Methods). Fractions were collected and separated by SDS-PAGE followed by western blotting with antibodies against the indicated proteins. In parallel, the lipid raft fraction (15%+20%+25% sucrose) was collected, immunoprecipitated with anti-CD59 antibody (B and C), separated by SDS-PAGE, and immunoblotted with anti-CD59 and anti-Myc antibodies (B) or with anti-CD59 and HA antibodies (C). IgGL: light chain of immunglobulins. (D)Untransfected cells [to detect endogenous CD59, transferrin receptor (TfR), ERGIC-53, and BAP- 31] or cells transfected with Myc-p23 or HA-p24 were pulsed for 10 minutes with [35S]methionine-cysteine and subjected to sucrose density gradient centrifugation (as in A). Fractions were immunoprecipitated with antibodies against the indicated proteins or Myc and HA, and separated by SDS-PAGE followed by autoradiography. 40+endo-H, immunoprecipitated CD59 and TfR were digested with endo-H (+endo-H) before analysis. (E)Lipid raft fractions (15+20+25) from 15-minute pulse-labeled cells were pooled, immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. Note co-immunoprecipitation with CD59. Positions of molecular mass markers (in kDa) are shown on the left.

    Techniques Used: Transfection, Gradient Centrifugation, SDS Page, Western Blot, Immunoprecipitation, Autoradiography, Labeling

    Fig. 7. Cholesterol depletion by MCD disrupts the interaction of GPI- anchored proteins and p24-p23. HeLa cells either untransfected (A,C,D) or transfected with Myc-p23 or with HA-p24 (B) or with glycosylated retrieval- impaired Myc-ERGIC-53 (Myc-ERGIC-53) (C,D) were treated (+) or not (–) with 20 mM MCD for 30 minutes at 37°C. (A)Cells were lysed in MBS- Triton X-100 at 4°C, run through 5-40% sucrose gradients, subjected to SDS- PAGE and immunoblotted with antibodies against the indicated proteins. (B)Cells were lysed in 1% Triton X-100 and immunoprecipitated with anti- CD59. The immunoprecipitates were separated by SDS-PAGE and western blotting was performed with anti-CD59, anti-Myc or anti-HA. IgGL, light chain of immunoglobulins. (C,D)Cells were pulsed with [35S]methionine- cysteine for 10 minutes, chased for 30 minutes (for CD59), 40 minutes (for transferrin-receptor) or 60 minutes (for Myc-ERGIC-53), lysed in 1% Triton X-100, and subjected to immunoprecipitation with antibodies against CD59, Myc or transferrin receptor. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, transferrin receptor (TfR) and Myc-ERGIC-53 (Myc-ERGIC-53) are indicated. MCD, methyl-– cyclodextrin.
    Figure Legend Snippet: Fig. 7. Cholesterol depletion by MCD disrupts the interaction of GPI- anchored proteins and p24-p23. HeLa cells either untransfected (A,C,D) or transfected with Myc-p23 or with HA-p24 (B) or with glycosylated retrieval- impaired Myc-ERGIC-53 (Myc-ERGIC-53) (C,D) were treated (+) or not (–) with 20 mM MCD for 30 minutes at 37°C. (A)Cells were lysed in MBS- Triton X-100 at 4°C, run through 5-40% sucrose gradients, subjected to SDS- PAGE and immunoblotted with antibodies against the indicated proteins. (B)Cells were lysed in 1% Triton X-100 and immunoprecipitated with anti- CD59. The immunoprecipitates were separated by SDS-PAGE and western blotting was performed with anti-CD59, anti-Myc or anti-HA. IgGL, light chain of immunoglobulins. (C,D)Cells were pulsed with [35S]methionine- cysteine for 10 minutes, chased for 30 minutes (for CD59), 40 minutes (for transferrin-receptor) or 60 minutes (for Myc-ERGIC-53), lysed in 1% Triton X-100, and subjected to immunoprecipitation with antibodies against CD59, Myc or transferrin receptor. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, transferrin receptor (TfR) and Myc-ERGIC-53 (Myc-ERGIC-53) are indicated. MCD, methyl-– cyclodextrin.

    Techniques Used: Transfection, SDS Page, Immunoprecipitation, Western Blot, Autoradiography

    Fig. 8. Effect of cholesterol depletion by MCD on the localization of endogenous CD59, ERGIC-53, p24 and Sec24C. HeLa cells were untreated (A-D) or treated (E-H) with 20 mM MCD for 30 minutes at 37°C and analyzed by immunofluorescence microscopy using antibodies against CD59, ERGIC-53 p24 and Sec24C. Note that CD59 and p24 localize to the ER in MCD-treated cells, whereas ERGIC-53 does not redistribute to the ER. Scale bar: 10m.
    Figure Legend Snippet: Fig. 8. Effect of cholesterol depletion by MCD on the localization of endogenous CD59, ERGIC-53, p24 and Sec24C. HeLa cells were untreated (A-D) or treated (E-H) with 20 mM MCD for 30 minutes at 37°C and analyzed by immunofluorescence microscopy using antibodies against CD59, ERGIC-53 p24 and Sec24C. Note that CD59 and p24 localize to the ER in MCD-treated cells, whereas ERGIC-53 does not redistribute to the ER. Scale bar: 10m.

    Techniques Used: Immunofluorescence, Microscopy



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    igg2a against human cd59  (Bio-Rad)
    96
    Bio-Rad igg2a against human cd59
    Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein <t>CD59</t> in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).
    Igg2a Against Human Cd59, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg2a against human cd59/product/Bio-Rad
    Average 96 stars, based on 1 article reviews
    igg2a against human cd59 - by Bioz Stars, 2026-03
    96/100 stars
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    Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein CD59 in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).

    Journal: Journal of cell science

    Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

    doi: 10.1242/jcs.062950

    Figure Lengend Snippet: Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein CD59 in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).

    Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

    Techniques: Transfection, Immunoprecipitation, SDS Page, Autoradiography, Pulse Chase, Control

    Fig. 3. CD59 interacts with p24 and p23. HeLa cells were either untransfected (–) or transfected (+) with Myc-p23 (A,B,E), Myc- p23 and CD59-GFP (C), HA-p24 (D,F), HA-p24 and CD59-GFP (D), as indicated. After 48 hours the cells were subjected to immunoprecipitation (Ip), SDS-PAGE, and western blotting (Wb) using the indicated antibodies. (E,F)Cells were pulsed with [35S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc- p23 and HA-p24 pulled down newly synthesized endogenous CD59 and vice versa. *Bands that probably correspond to endogenous p24 and p23. IgGH, heavy chain of immunoglobulins; IgGL, light chain of immunoglobulins. Positions of molecular mass markers are indicated on the left.

    Journal: Journal of cell science

    Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

    doi: 10.1242/jcs.062950

    Figure Lengend Snippet: Fig. 3. CD59 interacts with p24 and p23. HeLa cells were either untransfected (–) or transfected (+) with Myc-p23 (A,B,E), Myc- p23 and CD59-GFP (C), HA-p24 (D,F), HA-p24 and CD59-GFP (D), as indicated. After 48 hours the cells were subjected to immunoprecipitation (Ip), SDS-PAGE, and western blotting (Wb) using the indicated antibodies. (E,F)Cells were pulsed with [35S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc- p23 and HA-p24 pulled down newly synthesized endogenous CD59 and vice versa. *Bands that probably correspond to endogenous p24 and p23. IgGH, heavy chain of immunoglobulins; IgGL, light chain of immunoglobulins. Positions of molecular mass markers are indicated on the left.

    Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

    Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Autoradiography, Synthesized

    Fig. 6. Co-partitioning of GPI-anchored proteins and p24-p23 into lipid rafts. HeLa cells were untransfected (A) or were transfected with Myc-p23 (B) or with HA-p24 (C). Cells were lysed in MBS-Triton X-100 at 4°C and analyzed by sucrose gradient centrifugation (see Materials and Methods). Fractions were collected and separated by SDS-PAGE followed by western blotting with antibodies against the indicated proteins. In parallel, the lipid raft fraction (15%+20%+25% sucrose) was collected, immunoprecipitated with anti-CD59 antibody (B and C), separated by SDS-PAGE, and immunoblotted with anti-CD59 and anti-Myc antibodies (B) or with anti-CD59 and HA antibodies (C). IgGL: light chain of immunglobulins. (D)Untransfected cells [to detect endogenous CD59, transferrin receptor (TfR), ERGIC-53, and BAP- 31] or cells transfected with Myc-p23 or HA-p24 were pulsed for 10 minutes with [35S]methionine-cysteine and subjected to sucrose density gradient centrifugation (as in A). Fractions were immunoprecipitated with antibodies against the indicated proteins or Myc and HA, and separated by SDS-PAGE followed by autoradiography. 40+endo-H, immunoprecipitated CD59 and TfR were digested with endo-H (+endo-H) before analysis. (E)Lipid raft fractions (15+20+25) from 15-minute pulse-labeled cells were pooled, immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. Note co-immunoprecipitation with CD59. Positions of molecular mass markers (in kDa) are shown on the left.

    Journal: Journal of cell science

    Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

    doi: 10.1242/jcs.062950

    Figure Lengend Snippet: Fig. 6. Co-partitioning of GPI-anchored proteins and p24-p23 into lipid rafts. HeLa cells were untransfected (A) or were transfected with Myc-p23 (B) or with HA-p24 (C). Cells were lysed in MBS-Triton X-100 at 4°C and analyzed by sucrose gradient centrifugation (see Materials and Methods). Fractions were collected and separated by SDS-PAGE followed by western blotting with antibodies against the indicated proteins. In parallel, the lipid raft fraction (15%+20%+25% sucrose) was collected, immunoprecipitated with anti-CD59 antibody (B and C), separated by SDS-PAGE, and immunoblotted with anti-CD59 and anti-Myc antibodies (B) or with anti-CD59 and HA antibodies (C). IgGL: light chain of immunglobulins. (D)Untransfected cells [to detect endogenous CD59, transferrin receptor (TfR), ERGIC-53, and BAP- 31] or cells transfected with Myc-p23 or HA-p24 were pulsed for 10 minutes with [35S]methionine-cysteine and subjected to sucrose density gradient centrifugation (as in A). Fractions were immunoprecipitated with antibodies against the indicated proteins or Myc and HA, and separated by SDS-PAGE followed by autoradiography. 40+endo-H, immunoprecipitated CD59 and TfR were digested with endo-H (+endo-H) before analysis. (E)Lipid raft fractions (15+20+25) from 15-minute pulse-labeled cells were pooled, immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. Note co-immunoprecipitation with CD59. Positions of molecular mass markers (in kDa) are shown on the left.

    Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

    Techniques: Transfection, Gradient Centrifugation, SDS Page, Western Blot, Immunoprecipitation, Autoradiography, Labeling

    Fig. 7. Cholesterol depletion by MCD disrupts the interaction of GPI- anchored proteins and p24-p23. HeLa cells either untransfected (A,C,D) or transfected with Myc-p23 or with HA-p24 (B) or with glycosylated retrieval- impaired Myc-ERGIC-53 (Myc-ERGIC-53) (C,D) were treated (+) or not (–) with 20 mM MCD for 30 minutes at 37°C. (A)Cells were lysed in MBS- Triton X-100 at 4°C, run through 5-40% sucrose gradients, subjected to SDS- PAGE and immunoblotted with antibodies against the indicated proteins. (B)Cells were lysed in 1% Triton X-100 and immunoprecipitated with anti- CD59. The immunoprecipitates were separated by SDS-PAGE and western blotting was performed with anti-CD59, anti-Myc or anti-HA. IgGL, light chain of immunoglobulins. (C,D)Cells were pulsed with [35S]methionine- cysteine for 10 minutes, chased for 30 minutes (for CD59), 40 minutes (for transferrin-receptor) or 60 minutes (for Myc-ERGIC-53), lysed in 1% Triton X-100, and subjected to immunoprecipitation with antibodies against CD59, Myc or transferrin receptor. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, transferrin receptor (TfR) and Myc-ERGIC-53 (Myc-ERGIC-53) are indicated. MCD, methyl-– cyclodextrin.

    Journal: Journal of cell science

    Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

    doi: 10.1242/jcs.062950

    Figure Lengend Snippet: Fig. 7. Cholesterol depletion by MCD disrupts the interaction of GPI- anchored proteins and p24-p23. HeLa cells either untransfected (A,C,D) or transfected with Myc-p23 or with HA-p24 (B) or with glycosylated retrieval- impaired Myc-ERGIC-53 (Myc-ERGIC-53) (C,D) were treated (+) or not (–) with 20 mM MCD for 30 minutes at 37°C. (A)Cells were lysed in MBS- Triton X-100 at 4°C, run through 5-40% sucrose gradients, subjected to SDS- PAGE and immunoblotted with antibodies against the indicated proteins. (B)Cells were lysed in 1% Triton X-100 and immunoprecipitated with anti- CD59. The immunoprecipitates were separated by SDS-PAGE and western blotting was performed with anti-CD59, anti-Myc or anti-HA. IgGL, light chain of immunoglobulins. (C,D)Cells were pulsed with [35S]methionine- cysteine for 10 minutes, chased for 30 minutes (for CD59), 40 minutes (for transferrin-receptor) or 60 minutes (for Myc-ERGIC-53), lysed in 1% Triton X-100, and subjected to immunoprecipitation with antibodies against CD59, Myc or transferrin receptor. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, transferrin receptor (TfR) and Myc-ERGIC-53 (Myc-ERGIC-53) are indicated. MCD, methyl-– cyclodextrin.

    Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

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

    Fig. 8. Effect of cholesterol depletion by MCD on the localization of endogenous CD59, ERGIC-53, p24 and Sec24C. HeLa cells were untreated (A-D) or treated (E-H) with 20 mM MCD for 30 minutes at 37°C and analyzed by immunofluorescence microscopy using antibodies against CD59, ERGIC-53 p24 and Sec24C. Note that CD59 and p24 localize to the ER in MCD-treated cells, whereas ERGIC-53 does not redistribute to the ER. Scale bar: 10m.

    Journal: Journal of cell science

    Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

    doi: 10.1242/jcs.062950

    Figure Lengend Snippet: Fig. 8. Effect of cholesterol depletion by MCD on the localization of endogenous CD59, ERGIC-53, p24 and Sec24C. HeLa cells were untreated (A-D) or treated (E-H) with 20 mM MCD for 30 minutes at 37°C and analyzed by immunofluorescence microscopy using antibodies against CD59, ERGIC-53 p24 and Sec24C. Note that CD59 and p24 localize to the ER in MCD-treated cells, whereas ERGIC-53 does not redistribute to the ER. Scale bar: 10m.

    Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

    Techniques: Immunofluorescence, Microscopy