Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Complement factor staining in biopsies from kidney transplant recipients.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining, Blocking Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Antibodies for flow cytometric analysis on CiGEnCs.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques:

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: CD59 staining in vivo . Renal biopsy staining for complement regulator CD59 in glomeruli (A, C) and peritubular capillaries (B, D) . Staining was performed on human kidney before transplantation as control (A, B) , on biopsy specimen from a C4d-positive (C4d+) aABMR (C, D) . Black arrows point to CD59-positive peritubular capillaries, double-compound arrows to CD59-negative peritubular capillaries. aABMR, active antibody-mediated rejection.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining, In Vivo, Transplantation Assay

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Complement system activation on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis. Complement factors C3 (activated), C4d, and C5b-9 on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis are depicted in (A) with the four different incubation conditions plotted on the y-axis. Deposition of complement regulator, CD59, was measured in flow-cytometry under five different incubation conditions (B) . cABO, ABO-compatible; HLA, Human Leucocyte Antigen; Abs, antibodies; IgG, Immunoglobulin G; FITC, Fluorescein isothiocyanate; iABO, ABO-incompatible.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Activation Assay, In Vitro, Incubation, Flow Cytometry

Journal: Cell Division

Article Title: Complement susceptibility in glutamine deprived breast cancer cells

doi: 10.1186/1747-1028-2-20

Figure Lengend Snippet: Neutralization of CD59 enhanced complement-mediated lysis. In A through D, Bcap37 and MCF7 cells (1 × 10 5 ) were sensitized with 20 μg of rabbit antibody to β 2 -microglobulin (anti-β2M) with or without specified doses of mAb-YTH53.1 (anti-CD59). Background LDH activity was obtained with unsensitized cells similarly incubated with 25% NHS for 4.5 hours to provide a numerical value that was subtracted from experimental values. Experimental antibody combinations are represented as follows: 1) 10 μg anti-β2M alone (28.0% lysis ± 1.7). 2) 20 μg anti-β2M alone (42.3% lysis ± 2.9). 3) 20 μg mAb-YTH53.1 alone (11.3% lysis ± 1.5). 4) 20 μg anti-β2M + 5 μg mAb-YTH53.1 (43.3% lysis ± 2.1). 5) 20 μg anti-β2M + 10 μg mAb-YTH53.1 (53% lysis ± 4.8). 6) 20 μg anti-β2M + 20 μg mAb-YTH53.1 (59% lysis ± 4.9).

Article Snippet: Pellets were re-suspended in serum-free DMEM and aliquots of approximately 1 × 10 6 cells were probed with FITC-labeled monoclonal antibody to either CD55 (2 μg/ml mouse IgG1 BRIC-216, Serotec Inc.) or to CD59 (2 μg/ml mouse IgG2a, MEM43, Serotec Inc.).

Techniques: Neutralization, Lysis, Activity Assay, Incubation

Journal: Cell Division

Article Title: Complement susceptibility in glutamine deprived breast cancer cells

doi: 10.1186/1747-1028-2-20

Figure Lengend Snippet: Expression of CD59 and CD55 in breast cancer cells subjected to glutamine-mediated synchronization. Data is depicted as representative means for three independent experiments. Bcap37 cells (Figure 4a) and MCF7 cells (Figure 4b) were grown to confluency and either maintained with glutamine (Unsynchronized) or in the same media without glutamine for 48 hours (Glutamine Deprived), or in the same media without glutamine for 48 hours followed by 8 hours with glutamine (Glutamine Restored). In all experimental groups for Bcap37 and MCF7 cells, CD59 was quantified using FITC-conjugated anti-CD59 mouse monoclonal antibody and CD55 was quantified using FITC-conjugated anti-CD55 mouse monoclonal antibody.

Article Snippet: Pellets were re-suspended in serum-free DMEM and aliquots of approximately 1 × 10 6 cells were probed with FITC-labeled monoclonal antibody to either CD55 (2 μg/ml mouse IgG1 BRIC-216, Serotec Inc.) or to CD59 (2 μg/ml mouse IgG2a, MEM43, Serotec Inc.).

Techniques: Expressing

Journal: Cell Division

Article Title: Complement susceptibility in glutamine deprived breast cancer cells

doi: 10.1186/1747-1028-2-20

Figure Lengend Snippet: CD59 and CD55 expression in G0-G1 and G2-M subpopulations. Data is depicted as representative means for three independent experiments. Unsynchronized Bcap37 cells (Figure 5a) and MCF7 cells (Figure 5b) were grown to confluency and maintained in media supplemented with 2 mM L-glutamine (Unsynch). Synchronized cells were grown to confluency in normal media and then maintained in media without glutamine for 48 hours, followed by 8 hours in media supplemented with 2 mM L-glutamine (Gln Restored). Cells were then harvested, treated with propidium iodide, incubated with either FITC-conjugated antibodies to CD59 or to CD55 and prepared for flow cytometric sorting and analysis.

Article Snippet: Pellets were re-suspended in serum-free DMEM and aliquots of approximately 1 × 10 6 cells were probed with FITC-labeled monoclonal antibody to either CD55 (2 μg/ml mouse IgG1 BRIC-216, Serotec Inc.) or to CD59 (2 μg/ml mouse IgG2a, MEM43, Serotec Inc.).

Techniques: Expressing, Incubation

Journal: Cell Division

Article Title: Complement susceptibility in glutamine deprived breast cancer cells

doi: 10.1186/1747-1028-2-20

Figure Lengend Snippet: Percent Lysis following Glutamine Synchronization and Neutralization of CD59. Treatment groups were prepared as outlined for unsynchronized, glutamine deprived and glutamine restored populations of Bcap37 and MCF7 cells. Cells were sensitized with 20 μg of rabbit polyclonal antibody to β 2 -microglobulin and subjected to 25% fresh normal human serum (NHS) in combination with 20 μg of YTH53.1 rat monoclonal antibody to CD59. After 4 hours of incubation with NHS at 37°C, supernatants were collected and tested for LDH activity to determine percent lysis. Please note the increase in percent lysis for both cell lines as compared to Figure 6.

Article Snippet: Pellets were re-suspended in serum-free DMEM and aliquots of approximately 1 × 10 6 cells were probed with FITC-labeled monoclonal antibody to either CD55 (2 μg/ml mouse IgG1 BRIC-216, Serotec Inc.) or to CD59 (2 μg/ml mouse IgG2a, MEM43, Serotec Inc.).

Techniques: Lysis, Neutralization, Incubation, Activity Assay

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

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

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

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

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

Journal: Journal of Neuroinflammation

Article Title: Complement activation at the motor end-plates in amyotrophic lateral sclerosis

doi: 10.1186/s12974-016-0538-2

Figure Lengend Snippet: Primary antibodies and their dilutions

Article Snippet: CD59 , Mouse monoclonal , Anti-CD59 , Anti-human , 1:50 , HM2120/Hycult.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Complement activation at the motor end-plates in amyotrophic lateral sclerosis

doi: 10.1186/s12974-016-0538-2

Figure Lengend Snippet: Representative confocal triple-immunofluorescence for neurofilament (NF-H, Cy3), end-plates detected with α-BTX (Alexa 488) and the regulator CD59 (Cy5) in control ( a , b , c , d ) and ALS ( e , f , g , h ) intercostal muscle showing deposition of CD59 ( white asterisks in h , enlarged in insert ) in ALS intercostal muscle tissue on denervated end-plates ( white arrow pointing to α-BTX and arrowhead pointing to NF-H in h ) but not in controls. Quantification shows CD59-positive innervated and denervated motor end-plates in the intercostal muscle of ALS donors but not in controls ( P = 0.05 and P = 0.05, respectively) ( i ). Data represents standard deviation of the mean. n.d. not detected

Article Snippet: CD59 , Mouse monoclonal , Anti-CD59 , Anti-human , 1:50 , HM2120/Hycult.

Techniques: Immunofluorescence, Standard Deviation

Journal: Biochemistry and Biophysics Reports

Article Title: Induction of decay accelerating factor and membrane cofactor protein by resveratrol attenuates complement deposition in human coronary artery endothelial cells

doi: 10.1016/j.bbrep.2019.100652

Figure Lengend Snippet: Resveratrol increases MCP expression via a HO-1 dependent mechanism. (A) HCAECs were treated with resveratrol (0.001 μΜ) for 18 h and (B) HCAECs were transfected with 100 nM negative control (NC) or HO-1 siRNA for 48 h prior to resveratrol (0.001 μΜ) treatment for 18 h. MCP and CD59 proteins were analysed by immunoblotting and quantified by densitometry. Data are expressed as mean ± SEM (n = 3). β-actin was used as a loading control. *p < 0.05 vs no resveratrol, **p < 0.01 vs NC.

Article Snippet: Anti-DAF (clone 1C6, catalog number HM2280), anti-MCP (clone M177, catalog number HM2103) anti-CD59 (clone MEM-43, catalog number HM2120), anti-rat C3/C3b (clone 2B10B9B2; catalog number HM3031) and anti-human C3/C3b (clone 755, catalog number: HM2072) antibodies were purchased from Hycult (Uden, Netherlands).

Techniques: Expressing, Transfection, Negative Control, Western Blot

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Cell membrane expression of complement inhibitory protein (CIP) molecules [membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59] as assessed by flow cytometry of human lung cancer cell lines (non-small cell bronchogenic carcinoma) and of normal nasal epithelial cells in primary cultures.

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Effect of neutralizing antibodies against cell membrane complement inhibitory protein (CIP) molecules [anti-membrane cofactor protein (MCP), GB-24, decay-accelerating factor (DAF), IA-10, anti-CD59, YTH 53.1 or antiserum as indicated] on complement-mediated cell lysis of lung cancer cell lines (a) and of normal nasal epithelial cells (NEC) (b). Cells were incubated with antibodies (1:50 dilution, 30 min, at 4°C), then washed and exposed to 50% normal human serum (NHS). In some experiments cells were presensitized with anti-carcinoembryonic antigen (CEA) or with anti-NEC (1:20 dilution, each). Values are mean ± s.d. of four to six experiments. (a) *P < 0.01 versus lysis of same cells with NHS and no antibodies or versus lysis with anti-CD59; **P < 0.025 versus lysis of same cells with NHS and no antibodies. (b) **P < 0.001 versus lysis of same cells with NHS and no antibodies (Student's t-test for all comparisons). Data for normal NEC, except for the effect of anti-CD59, YTH 53.1, were obtained from our recent concomitant study [5].

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Lysis, Incubation

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Detachment of decay-accelerating factor (DAF) and CD59 from the NCI-H596 lung cancer cell line cell membrane by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC; 0.5 U/ml, 37°C, 45 min). Expression of cell membrane DAF and CD59 was assayed by flow cytometry. Results for ChaGo K-1 cells were the same (not shown).

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro , and an insight into mechanism(s) of resistance

doi: 10.1046/j.1365-2249.1998.00581.x

Figure Lengend Snippet: Effect of decay-accelerating factor (DAF) and CD59 detachment from the cell membrane of lung cancer cell lines on the cells’ susceptibility to complement-mediated lysis. (a) Cells were treated first with phosphatidylinositol-specific phospholipase C (PIPLC; 45 min, 37°C) at various concentrations, then washed and exposed to 50% normal human serum (NHS; 60 min, 37°C). *P < 0.001 from the alternate preceding value; **P < 0.05 from preceding value for ChaGo K-1 cells; ***P < 0.01 from the alternate preceding value, for NCI-H596 cells. (b) Cells were first treated with PIPLC, then washed and exposed to various concentrations of NHS. **P < 0.001 from the preceding value for both cell types. Values are mean ± s.d. of four to six experiments (one-way anova for all comparisons).

Article Snippet: Rat anti-human CD59 MoAb YTH 53.1 (IgG2b) was purchased from Serotec (Oxford, UK) [ 12 ].

Techniques: Lysis

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) Shaded (with solid black) residues are the amino acids that match the consensus sequence. Gaps introduced into sequences to optimize alignment are represented by dashes. The asterisk represents the ten cysteines residues that are conserved among members of the family. The residues enclosed in a box indicate the conserved CD59/Ly-6 family motif CCXXXXCN. (B) Alignments of gecko CD59 with pfam00021, the typical sequence of CD59 family. The conserved and active sites are marked by gray color and the predicted disulfide bonds are also indicated by lines. Sequences obtained from GenBank or SwissProt are gecko (HM208338), human (NP_000602.1), mouse-a (NP_031678), mouse-b (NP_862906), rat (AAH63176), trout 1 (AY593999), trout 2 (AJ880383), hagfish (AAD47892), Prod1 (ABV29331).

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) 293T cells transfected with pEGFP-N3 plasmids showing expression in cytoplasm and nucleus. (B) Colocalization of CD59 and DiI dye in the cell membrane at 7 days after transfection with pEGFP-N3-CD59 plasmids, counterstaining with the Hoechst 33342.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Transfection, Expressing, Membrane

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) Northern blotting analysis of CD59 transcripts in different gecko tissues. The bands indicate the position of molecular size equivalent to 2,100 bp. (B) Semi-quantitative RT-PCR analysis of CD59 transcripts in spinal cord along the anterior-posterior axis. Gecko EF-1α was used for normalization. C, cervical segment, from 1 st –8 th cervical vertebra; T, thoracic segment, from 1 st –5 th thoracic vertebra; L, lumbar segment, from 1 st –13 th lumbar vertebra; and S, sacral segment, from 1 st –2 nd sacral vertebra. (C) Expression of CD59 in spinal cord was analyzed by in situ hybridization. C1, T1, L1 and S1 are magnifications of C, T, L and S, respectively. Scale bars: C-S, 100 µm; C1-S1, 30 µm.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Northern Blot, Quantitative RT-PCR, Expressing, In Situ Hybridization

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) RT-PCR amplification of CD59 for four average segments of the tail. (B) Expression of CD59 in the tail was analyzed by in situ hybridization. Positive signals in the gray matter and ependymal cells are indicated on the micrographs.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, In Situ Hybridization

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) Quantitative results for RT-PCR amplification of CD59 for the spinal cord from L13 to the 6 th caudal vertebra for the controls (Nor) and following tail amputation at 1 day, 3 days, 1 week and 2 weeks. Quantities were normalized to endogenous EF-1α expression. Error bars represent the standard deviation (P<0.01). (B) Localization of CD59 mRNA in the spinal cord by in situ hybridization using CD59 antisense RNA probes. L, M, O and P indicate sections of spinal cord segments at 1 day, 3 days, 1 week and 2 weeks post amputation. J indicates control section with sense probe. Scale bars: J-P, 50 µm.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Standard Deviation, In Situ Hybridization, Control

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) Engulfment behavior of blastemal confrontation cultures. The time course of engulfment is shown by sequential images at 1 day to 6 days. The proximal blastema is labeled with a tracker DiI. (B), (C) and (D) Coculture in the presence of human CD59 antibodies at dilution 1∶1000, 1∶500 and 1∶250, respectively. (E) PD engulfment was blocked co-culturing with rabbit-anti human CD59 antibody at 1∶100. (F) Coculture of a proximal with a distal blastema in the presence of rabbit-anti human β-actin antibody at 1∶100. The β-actin antibody did not generate an inhibitory effect. (G) Cross reaction of rabbit-anti human CD59 antibody with gecko blastema was analyzed by western blot. (H) Occurrence frequency of the PD engulfment in A–F.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Labeling, Western Blot

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: Geckos were amputated and injected with DMSO (A–D) or RA (E–F). (A) showing the expression of CD59 in the blastemas at 1 week post amputation and (C) at 20 days post amputation; (B) and (D) indicating high power view of (A) and (C), respectively. (E) showing the expression of the CD59 in the blastems of RA-treated animals at 1 week post amputation and (F) 20 days post amputation. (G) RT-PCR analysis of CD59 transcripts in the 20-day blastemas of DMSO-treated or RA-treated animals. Each blastema was cut averagely prior to RNA extraction. Quantities were normalized to endogenous EF-1α expression. Error bars represent the standard deviation (P<0.01). Line marks the plane of amputation. P, proximal segment of blastema; D, distal segment of blastema, P-RA, proximal segment of blastema treated with RA; D-RA, distal segment of blastema treated with RA. Scale bars: A, C, E, F, 200 µm; B, D, 50 µm.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Standard Deviation

Journal: PLoS ONE

Article Title: Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration

doi: 10.1371/journal.pone.0017878

Figure Lengend Snippet: (A) 20-day blastema electroporated with pEGFP-N3-CD59 plasmid at 3 days post-electroporation. White circle indicates the plasmid-injected site; (B) Dorsal view of the (A); (C) Parallel experiment electroporated with pDsRed-monoter-C1 was used as a control. Dot line area indicates part of the blastema cut away for better capture of image.

Article Snippet: The membrane was then blocked with 5% non-fat dry milk in TBS containing 0.05% Tween-20 (TBS-T) for 1 h and incubated with polyclonal rabbit anti-human CD59 IgG (ProteinTech Group, Inc. Chicago, 1∶1,500).

Techniques: Plasmid Preparation, Electroporation, Injection, Control