Journal: PLoS ONE

Article Title: Amyloid Properties of the Mouse Egg Zona Pellucida

doi: 10.1371/journal.pone.0129907

Figure Lengend Snippet: A) Thirty isolated ZP were treated with (+) or without (-) DMSO followed by incubation with the PAD ligand and eluted proteins examined by immunoblot using anti-ZP1, ZP2, and ZP3 antibodies. ZP, an equivalent number of ZP placed directly in SDS-PAGE loading buffer without PAD incubation. Buffer, PBS only incubated with PAD beads. Molecular weight markers indicate kDa. B) phase images of oocytes incubated in the presence (+) or absence (no DMSO) of 90% DMSO for 2 minutes at room temperature. Arrow indicates ZP. Scale bar = 50 μm. C) Equal numbers of ZP were exposed to 90% DMSO, 1% SDS or 0.25% SDS prior to spotting on nitrocellulose using a dot blot apparatus. Membranes were then incubated with the amyloid anti-fibrillar OC and anti-oligomer A11 antibodies. Dot blots were rehybridized with anti-ZP2 antibody to confirm the presence of ZP protein in each well. b, buffer.

Article Snippet: Samples were electro blotted onto a polyvinylidene difluoride membrane (cat. no. IPVH00010, EMD Millipore, Bedford, MA) and the membrane was blocked for 1 h at RT with shaking in 3% nonfat dry milk in TBST and then incubated overnight with shaking at 4°C with either 0.5 μg/ml rat anti-mouse ZP1 (M1.4, sc-32751, Santa Cruz Biotechnology, Santa Cruz, CA), 0.25 μg/ml rat anti-mouse ZP2 (IE-3, sc-32752, Santa Cruz Biotechnology), or 0.5 μg/ml rabbit anti-human ZP3 (H-300, sc-25802, Santa Cruz Biotechnology) in 3% nonfat dry milk in TBST.

Techniques: Isolation, Incubation, Western Blot, SDS Page, Molecular Weight, Dot Blot

Journal: PLoS ONE

Article Title: Amyloid Properties of the Mouse Egg Zona Pellucida

doi: 10.1371/journal.pone.0129907

Figure Lengend Snippet: A) Schematic diagram of mature ZP1, ZP2, and ZP3 with amyloidogenic regions predicted by AmylPred 2 indicated as red bars above the individual domains. Yellow box, trefoil domain. Numbers signify amino acid number. B) Structure based sequence alignment of the ZP polymerization domain of mouse ZP1 (aa 268–541), ZP2 (aa 361–630), and ZP3 (aa 42–305) showing amyloidogenic sites (blue highlighting), as predicted by the Amylpred2 algorithm. Cysteine residues are noted by black boxes. The internal hydrophobic patch (IHP) is indicated by a red box . The β-strand secondary structure based on the crystal structure of chicken ZP3 is noted by orange bars (ZP-N subdomain) and green bars (ZP-C subdomain) above the amino acid sequences . C) Structure based alignment of ZP-N domains in mouse ZP1 (N1, N2), ZP2 (N1-N4), ZP3 (N), abalone VERL repeat 10 (R10) and yeast α-agglutinin/Sag 1p showing predicted amyloidogenic sites in blue highlighting as determined by the AmylPred2 algorithm. The β-strand secondary structure, based on structure of mZP-N is indicated by orange bars above the amino acid sequence . Cysteine residues are noted by black boxes. * , indicate sites essential or important for ZP2-sperm and α-agglutinin-a-agglutinin binding [ , ].

Article Snippet: Samples were electro blotted onto a polyvinylidene difluoride membrane (cat. no. IPVH00010, EMD Millipore, Bedford, MA) and the membrane was blocked for 1 h at RT with shaking in 3% nonfat dry milk in TBST and then incubated overnight with shaking at 4°C with either 0.5 μg/ml rat anti-mouse ZP1 (M1.4, sc-32751, Santa Cruz Biotechnology, Santa Cruz, CA), 0.25 μg/ml rat anti-mouse ZP2 (IE-3, sc-32752, Santa Cruz Biotechnology), or 0.5 μg/ml rabbit anti-human ZP3 (H-300, sc-25802, Santa Cruz Biotechnology) in 3% nonfat dry milk in TBST.

Techniques: Sequencing, Binding Assay

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet: Clinical characterization (A) Images of oocytes or COCs from the control and patient with the ZP2 heterozygous mutation. Scale bar, 50 μm. (B) The morphology of early embryogenesis from ICSI-treated MII oocytes from the control and proband. Scale bar, 50 μm.

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Control, Mutagenesis

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet: The heterozygous missense mutation in ZP2 was identified (A) Pedigree of the ZP2 pathogenic mutation. Square represented male, circle represented female, solid circle represented affected members, equal sign represented infertility, and the arrow indicated the proband, WT represented wild type. (B) Sanger sequencing results of ZP2 gene from the patient and her family members. (C) Location and conservation of the mutation in ZP2 protein. ZP2 contains 736 amino acids, including signal peptide (SP), ZP domain (ZPD) consisting of ZP-N and ZP-C subdomains, consensus furin cleavage site (CFCS), internal hydrophobic peptide (IHP), external hydrophobic peptide (EHP), and transmembrane domain (TMD). The red arrow indicated the position of mutated amino acid and the Thr539 was highly conserved in multiple species. (D) Structure prediction of ZP2 WT and ZP2 T539M protein by PyMOL software. The overall structure of ZP2-C subdomain was shown on the left, with the black box representing the region of the mutation site. The enlarged structure, on the right, displayed interaction of Thr539/Met539 with surrounding amino acids. Red represented α-helixes, yellow represented β-sheets, and green represented loops. Black dashed line represented hydrogen bonds.

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Mutagenesis, Sequencing, Software

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet: The missense mutation caused reduced ZP2 expression and secretion (A) The ZP2 of cell lysate and media in 293T cells transfected with ZP2 WT , ZP2 WT/T539M , and ZP2 T539M vectors by western blotting. The molecular weight of ZP2 was 100 kDa, and the molecular weight of β-actin was 42 kDa. (B) Statistical analysis of relative ZP2 level in cell lysate of each group. (C) Statistical analysis of relative ZP2 level in media of each group. (D) Localization of ZP2 in 293T cells transfected with ZP2 WT , ZP2 WT/T539M , and ZP2 T539M vectors. Green, ZP2; blue, DNA; scale bar, 10 μm. (E) ZP2 immunofluorescent intensity in three groups. All experiments were repeated at least three times. Data were presented as mean ± SEM, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Mutagenesis, Expressing, Transfection, Western Blot, Molecular Weight

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet: Zp2 T541M female mice were infertile (A) Schematic diagram of the preparation of Zp2 mutant mice using CRISPR-Cas9 technology. c.1622-1623CT>TG located in exon14 was the missense mutation. c.1632G>A was a deliberately introduced synonymous mutation. SsDNA: single-stranded DNA; sgRNA: single-guide RNA; HR: homologous recombination. (B) Gene sequences of Zp2 WT , Zp2 WT/T541M , and Zp2 T541M mice were identified by Sanger sequencing. The bases in the red box were the mutant. (C) Fertility curves showed the total number of litters per female for 6 months. At least 3 female mice of each genotype were used in the experiment. (D) The numbers of MII oocytes in female mice of three groups after superovulation treatment. (E)The bright-field images from superovulated Zp2 WT , Zp2 WT/T541M and Zp2 T541M MII oocytes of 6-week-old mice. Bar, 100 μm. (F) ZP thickness of MII oocytes for each group. All experiments were repeated at least three times. Data were presented as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Mutagenesis, CRISPR, Homologous Recombination, Sequencing

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet: Zp2 T541M mutation interfered with ZP2 secretion and ZP formation (A) PAS staining images of ovarian sections in 2-month-old Zp2 WT , Zp2 WT/T541M , and Zp2 T541M female mice. Bar, 100 μm. (B) Immunofluorescent staining of ZP2 in 2-month-old Zp2 WT , Zp2 WT/T541M , and Zp2 T541M ovarian sections. Green, ZP2; blue, DNA; bar, 100 μm. (C) Western blot analysis of ZP2 in cell lysate and media of 3T3 cells transfected with Zp2 WT , Zp2 WT/T541M , and Zp2 T541M vectors. The molecular weight of ZP2 was 90 kDa, and the molecular weight of β-actin was 42 kDa. (D) Statistical analysis of relative ZP2 expression level in cell lysate of each group. (E) Statistical analysis of relative ZP2 secretion level in media of each group. All experiments were repeated at least three times. Data were presented as mean ± SEM,∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Mutagenesis, Staining, Western Blot, Transfection, Molecular Weight, Expressing

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet: Defected folliculogenesis in Zp2 T541M female mice (A) H&E staining of ovarian sections in 2-month-old Zp2 WT , Zp2 WT/T541M , and Zp2 T541M female mice. Bar, 100 μm. (B) Quantification of follicles at all stages in ovaries from three groups. At least three female mice of each genotype were used. Data were presented as mean ± SEM, ∗p < 0.05. (C) Phalloidin staining of frozen ovarian sections from 2-month-old female mice. Green, Phalloidin; blue, DAPI; white arrowheads, TZPs; bar, 20 μm. (D) Ovarian paraffin sections from 2-month-old female mice were stained with TUNEL. Green, TUNEL; blue, DAPI; bar, 100 μm.

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Staining, TUNEL Assay

Journal: iScience

Article Title: A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

doi: 10.1016/j.isci.2023.107828

Figure Lengend Snippet:

Article Snippet: Anti-ZP2 antibody , Boster , Cat#PB0958;.

Techniques: Recombinant, Lysis, Staining, Bicinchoninic Acid Protein Assay, TUNEL Assay, Mutagenesis, Software, Sequencing

Journal: Cancers

Article Title: Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment

doi: 10.3390/cancers15072171

Figure Lengend Snippet: Six of the potential markers are robustly translated into protein within KS tumors. ( A – F ) Dual IF of the co-expression of LANA and the indicated potential KS biomarker. FLT4, KDR, UNC5A, ADAM12, CD34, and Prox-1 had robust expression in the KS lesions and were not exclusively detected in LANA-positive cells. ZP2 and OX40 were not detected at the protein level in KS lesions despite high transcriptomic expression. LANA is denoted as red, and the surface marker in question is green. Open yellow arrows indicate LANA-positive cells, and closed arrows indicate LANA-negative cells.

Article Snippet: The primary antibodies used are as follows: rat anti-LANA (Abcam, 1:100), mouse-anti CD34 (Invitrogen, 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, 1:200), rabbit-anti FLT4 (Invitrogen, 1:100), rabbit-anti ADAM12 (Invitrogen, 1:100), rabbit-anti UNC5A (ProteinTech, 1:100), rabbit-anti OX40 (Novus, 1:100), and rabbit-anti ZP2 (Invitrogen, 1:100).

Techniques: Expressing, Biomarker Discovery, Marker

Journal: Cancers

Article Title: Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment

doi: 10.3390/cancers15072171

Figure Lengend Snippet: Dual IF of KSHV-infected L1T2 and uninfected TIVE cells shows the expression of KDR, FLT4, and UNC5a but not ADAM12 or the endothelial markers. ( A – L ) Dual IF of the co-expression of LANA and the indicated potential KS biomarker. ADAM12, CD34, and Prox-1 were not detected in either cell line. FLT4, KDR, and UNC5A had low to moderate expression in the KS lesions and were not exclusively detected in LANA-positive cells. LANA is denoted as red, and the surface marker in question is green. Open yellow arrows indicate LANA-positive cells, and closed arrows indicate LANA-negative cells. Images were acquired at 20X magnification.

Article Snippet: The primary antibodies used are as follows: rat anti-LANA (Abcam, 1:100), mouse-anti CD34 (Invitrogen, 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, 1:200), rabbit-anti FLT4 (Invitrogen, 1:100), rabbit-anti ADAM12 (Invitrogen, 1:100), rabbit-anti UNC5A (ProteinTech, 1:100), rabbit-anti OX40 (Novus, 1:100), and rabbit-anti ZP2 (Invitrogen, 1:100).

Techniques: Infection, Expressing, Biomarker Discovery, Marker

Journal: Cancers

Article Title: Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment

doi: 10.3390/cancers15072171

Figure Lengend Snippet: Dual IF demonstrating the colocalization of markers and LANA in mouse xenograft tissue. ( A – F ) Dual IF of the co-expression of LANA and the indicated potential KS biomarker. FLT4, KDR, UNC5A, and ADAM12 had robust expression in the KS lesions and were not exclusively detected in LANA-positive cells. CD34 and Prox-1 were not detected at the protein level. LANA is denoted as red, and the surface marker in question is green. Images were acquired at 20X magnification.

Article Snippet: The primary antibodies used are as follows: rat anti-LANA (Abcam, 1:100), mouse-anti CD34 (Invitrogen, 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, 1:200), rabbit-anti FLT4 (Invitrogen, 1:100), rabbit-anti ADAM12 (Invitrogen, 1:100), rabbit-anti UNC5A (ProteinTech, 1:100), rabbit-anti OX40 (Novus, 1:100), and rabbit-anti ZP2 (Invitrogen, 1:100).

Techniques: Expressing, Biomarker Discovery, Marker

Journal: Cancers

Article Title: Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment.

doi: 10.3390/cancers15072171

Figure Lengend Snippet: Figure 3. Six of the potential markers are robustly translated into protein within KS tumors. (A–F) Dual IF of the co-expression of LANA and the indicated potential KS biomarker. FLT4, KDR, UNC5A, ADAM12, CD34, and Prox-1 had robust expression in the KS lesions and were not exclusively detected in LANA-positive cells. ZP2 and OX40 were not detected at the protein level in KS lesions despite high transcriptomic expression. LANA is denoted as red, and the surface marker in question is green. Open yellow arrows indicate LANA-positive cells, and closed arrows indicate LANA-negative cells.

Article Snippet: The antibodies used for IHC were as follows: mouse anti-LANA (Leica, Deer Park, IL, USA, 1:100), rat anti-LANA (Abcam, Boston, MA, USA, 1:100), mouse-anti CD34 (Invitrogen, Boston, MA, USA 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, Rosemont, IL, USA, 1:1500), rabbit-anti FLT4 (Invitrogen, 1:800), rabbit-anti ADAM12 (Invitrogen, 1:500), rabbit-anti UNC5A (ProteinTech, 1:300), rabbit-anti OX40 (Novus, Centennial, CO, USA 1:500), rabbit-anti ZP2 (Invitrogen, 1:5000).

Techniques: Expressing, Biomarker Discovery, Marker

Journal: Cancers

Article Title: Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment.

doi: 10.3390/cancers15072171

Figure Lengend Snippet: Figure 6. Dual IF of KSHV-infected L1T2 and uninfected TIVE cells shows the expression of KDR, FLT4, and UNC5a but not ADAM12 or the endothelial markers. (A–L) Dual IF of the co-expression of LANA and the indicated potential KS biomarker. ADAM12, CD34, and Prox-1 were not detected in either cell line. FLT4, KDR, and UNC5A had low to moderate expression in the KS lesions and were not exclusively detected in LANA-positive cells. LANA is denoted as red, and the surface marker in question is green. Open yellow arrows indicate LANA-positive cells, and closed arrows indicate LANA-negative cells. Images were acquired at 20X magnification.

Article Snippet: The antibodies used for IHC were as follows: mouse anti-LANA (Leica, Deer Park, IL, USA, 1:100), rat anti-LANA (Abcam, Boston, MA, USA, 1:100), mouse-anti CD34 (Invitrogen, Boston, MA, USA 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, Rosemont, IL, USA, 1:1500), rabbit-anti FLT4 (Invitrogen, 1:800), rabbit-anti ADAM12 (Invitrogen, 1:500), rabbit-anti UNC5A (ProteinTech, 1:300), rabbit-anti OX40 (Novus, Centennial, CO, USA 1:500), rabbit-anti ZP2 (Invitrogen, 1:5000).

Techniques: Infection, Expressing, Biomarker Discovery, Marker

Journal: Cancers

Article Title: Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment.

doi: 10.3390/cancers15072171

Figure Lengend Snippet: Figure 7. Dual IF demonstrating the colocalization of markers and LANA in mouse xenograft tissue. (A–F) Dual IF of the co-expression of LANA and the indicated potential KS biomarker. FLT4, KDR, UNC5A, and ADAM12 had robust expression in the KS lesions and were not exclusively detected in LANA-positive cells. CD34 and Prox-1 were not detected at the protein level. LANA is denoted as red, and the surface marker in question is green. Images were acquired at 20X magnification.

Article Snippet: The antibodies used for IHC were as follows: mouse anti-LANA (Leica, Deer Park, IL, USA, 1:100), rat anti-LANA (Abcam, Boston, MA, USA, 1:100), mouse-anti CD34 (Invitrogen, Boston, MA, USA 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, Rosemont, IL, USA, 1:1500), rabbit-anti FLT4 (Invitrogen, 1:800), rabbit-anti ADAM12 (Invitrogen, 1:500), rabbit-anti UNC5A (ProteinTech, 1:300), rabbit-anti OX40 (Novus, Centennial, CO, USA 1:500), rabbit-anti ZP2 (Invitrogen, 1:5000).

Techniques: Expressing, Biomarker Discovery, Marker