ProSci

PDL-2 Antibody

Product Code:
 
PSI-4063
Product Group:
 
Primary Antibodies
Supplier:
 
ProSci
Host Type:
 
Rabbit
Antibody Isotype:
 
IgG
Antibody Clonality:
 
Polyclonal
Regulatory Status:
 
RUO
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunofluorescence (IF)
  • Immunohistochemistry (IHC)
  • Western Blot (WB)
1 / 10
<strong>Figure 1 Western Blot Validation in Human Raji Cell Lysate</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PD-L2 4063 (A: 0.5 μg/mL and B: 1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
2 / 10
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Human and Mouse Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PD-L2, 4063 (4 μg/mL), competitor antibody (4 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
3 / 10
<strong>Figure 3 KO Validation in HeLa Cells</strong><br> Loading: 15 μg of HeLa WT cell lysates or PD-L2 KO cell lysates. Antibodies: PD-L2, 4063 (4 μg/mL) and beta-actin 3779 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
4 / 10
<strong>Figure 3 KO Validation in HeLa Cells</strong><br> Loading: 15 μg of HeLa WT cell lysates or PD-L2 KO cell lysates. Antibodies: PD-L2, 4063 (4 μg/mL) and beta-actin 3779 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
5 / 10
<strong>Figure 5 Immunohistochemistry Validation of PD-L2 in Mouse Brain Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-PD-L2 antibody (4063) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
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<strong>Figure 6 Immunofluorescence Validation of PD-L2 in Mouse Brain Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain cells labeling PD-L2 with 4063 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
7 / 10
<strong>Figure 7 Immunohistochemistry Validation of PD-L2 in Mouse Brain Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-PD-L2 antibody (4063) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
8 / 10
<strong>Figure 8 Immunofluorescence Validation of PD-L2 in Mouse Brain Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain tissue labeling PD-L2 with 4063 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
9 / 10
<strong>Figure 9 Immunohistochemistry Validation of PD-L2 in Lung Tumor of Mice (Kao et al., 2015) </strong><br> Protein analysis for PD-L2 (E-H) by immunohistochemistry with anti-PD-L2 antibodies in mice lung tumors. hMUC1.Tg mice were induced with lung adenoma and then treated with concurrent or sequential cisplatin/radiotherapy. PD-L2 expression level at week 41 after treatment was similar in control and treatment groups.
10 / 10
<strong>Figure 10 Regulated Expression Validation of PD-L2 in Mice with Melanoma Tumor (Knox et al., 2019) </strong><br> Immunoblot analysis of PD-L2 expression with anti-PD-L2 (4063) antibodies. PD-L2 expression was up-regulated by anti-PD1 antibody treatment whereas it was reduced by Next A alone or combination treatment (anti-PD1 antibody + NextA).
❮❯

<strong>Figure 1 Western Blot Validation in Human Raji Cell Lysate</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PD-L2 4063 (A: 0.5 μg/mL and B: 1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Human and Mouse Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PD-L2, 4063 (4 μg/mL), competitor antibody (4 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 KO Validation in HeLa Cells</strong><br> Loading: 15 μg of HeLa WT cell lysates or PD-L2 KO cell lysates. Antibodies: PD-L2, 4063 (4 μg/mL) and beta-actin 3779 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 KO Validation in HeLa Cells</strong><br> Loading: 15 μg of HeLa WT cell lysates or PD-L2 KO cell lysates. Antibodies: PD-L2, 4063 (4 μg/mL) and beta-actin 3779 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunohistochemistry Validation of PD-L2 in Mouse Brain Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-PD-L2 antibody (4063) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 6 Immunofluorescence Validation of PD-L2 in Mouse Brain Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain cells labeling PD-L2 with 4063 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
<strong>Figure 7 Immunohistochemistry Validation of PD-L2 in Mouse Brain Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-PD-L2 antibody (4063) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 8 Immunofluorescence Validation of PD-L2 in Mouse Brain Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain tissue labeling PD-L2 with 4063 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
<strong>Figure 9 Immunohistochemistry Validation of PD-L2 in Lung Tumor of Mice (Kao et al., 2015) </strong><br> Protein analysis for PD-L2 (E-H) by immunohistochemistry with anti-PD-L2 antibodies in mice lung tumors. hMUC1.Tg mice were induced with lung adenoma and then treated with concurrent or sequential cisplatin/radiotherapy. PD-L2 expression level at week 41 after treatment was similar in control and treatment groups.
<strong>Figure 10 Regulated Expression Validation of PD-L2 in Mice with Melanoma Tumor (Knox et al., 2019) </strong><br> Immunoblot analysis of PD-L2 expression with anti-PD-L2 (4063) antibodies. PD-L2 expression was up-regulated by anti-PD1 antibody treatment whereas it was reduced by Next A alone or combination treatment (anti-PD1 antibody + NextA).

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Further Information

Additional Names:
PD-L2 Antibody: B7DC, Btdc, PDL2, CD273, PD-L2, PDCD1L2, bA574F11.2, B7DC, Programmed cell death 1 ligand 2, Butyrophilin B7-DC, PD-1 ligand 2
Application Note:
WB: 0.5-4 μg/mL; IHC: 2.5 μg/mL; IF: 20 μg/mL.

Antibody validated: Western Blot in human and mouse samples; Immunohistochemistry in mouse samples; Immunofluorescence in mouse samples. All other applications and species not yet tested.
Background:
PD-L2 Antibody: Cell-mediated immune responses are initiated by T lymphocytes that are themselves stimulated by co gnate peptides bound to MHC molecules on antigen-presenting cells (APC). T-cell activation is generally self-limited as activated T cells express receptors such as PD-1 (also known as PDCD-1) that mediate inhibitory signals from the APC. PD-1 can bind two different but related ligands, PD-L1 and PD-L2, both of which are thought act as a negative regulator of T cell activation. However, it has been suggested that PD-L2 can act to stimulate an immunogenic response through and alternative receptor from PD-1.
Background References:
  • Holling et al. Hum. Immunol. 2004; 65:282-90.
  • Ishida et al. EMBO J. 1992; 11:3887-95.
  • LaGier and Pober. Hum. Immunol. 2006; 67:568-78.
  • Zhang et al. Proc. Natl. Acad. Sci. USA 2006; 103:11695-700.
Buffer:
PD-L2 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Conjugate:
Unconjugated
DISCLAIMER:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.
Homology:
Predicted species reactivity based on immunogen sequence: rat (94%).
Immunogen:
Anti-PD-L2 antibody (4063) was raised against a peptide corresponding to 16 amino acids near the center of human PD-L2.

The immunogen is located within amino acids 140-190 of PD-L2.
ISOFORMS:
Human PD-L2 has 3 isoforms, including isoform 1 (273aa, 31kD), isoform 2 (183aa, 21kD) and isoform 3 (182aa, 21kD). Mouse PD-L2 has one isoform (247aa, 28kD) and Rat PD-L2 also has one isoform (268aa, 30kD). 4063 can detect human isoform and mouse isoform.
NCBI Gene ID #:
80380
NCBI Official Name:
programmed cell death 1 ligand 2
NCBI Official Symbol:
PDCD1LG2
NCBI Organism:
Homo sapiens
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
Predicted: 31kD

Observed: 29 kD
Protein Accession #:
NP_079515
Protein GI Number:
190014605
Purification:
PD-L2 Antibody is affinity chromatography purified via peptide column.
Research Area:
Apoptosis
Swissprot #:
Q9BQ51
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

Independent Antibody Validation in Cell lines (Figure 2) shows similar PD-L2 expression profile in human and mouse cell lines detected by two independent anti-PD-L2 antibodies that recognize different epitopes, 4063 against central domain and the competitor antibody against N-terminus domain.  PD-L2 proteins are detected in the most tested cell lines at different expression levels by the two independent antibodies. 

KO Validation (Figure 3): Anti-PD-L2 antibodies (4063) specificity was further verified by PD-L2 specific knockout. PD-L2 signal was not detected in PD-L2 knockout HeLa cells as compared to that in control wild type cells.

Recombinant Protein Test (Figure 4): Anti-PD-L2 antibodies (4063) detected human PD-L2 recombinant protein at different concentrations.

Regulated expression validation (Figure 11): PD-L2 expression detected by anit-PD-L2 antibodies (4063) was up-regulated by anti-PD1 antibody treatment, which was reduced by Next A alone or combination treatment (anti-PD1 antibody + NextA).

References

  1. Kao et al. Assessing the Effects of Concurrent versus Sequential Cisplatin/Radiotherapy on Immune Status in Lung Tumor-Bearing C57BL/6 Mice. Cancer Immunol Res. 2015;3(7):741-50. PMID: 25672395
  2. Knox et al. Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells. Sci Rep. 2019;9(1):6136. PMID: 30992475