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