PHAP I Antibody

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ProSci
Product Code: PSI-3145
Product Group: Primary Antibodies
Supplier: ProSci
CodeSizePrice
PSI-3145-0.02mg0.02mg£150.00
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PSI-3145-0.1mg0.1mg£449.00
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Overview

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)

Images

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<strong>Figure 1 Western Blot Validation in Human Raji Cell Lysate</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PHAP I 3145 (A: 2 μg/mL, B: 4 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PHAP I 3145 (2 μg/mL), PHAP I 3151 (1 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.
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<strong>Figure 3 Western Blot Validation in Human Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PHAP I 3145 (2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 4 Immunofluorescence Validation of PHAP I in Raji Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed Raji Cells labeling PHAP I with 3145 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
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<strong>Figure 5 Immunocytochemistry Validation of PHAP I in Raji Cells</strong><br> Immunocytochemical analysis of Raji cells using anti-PHAP I antibody (3145) at 2 μg/ml. Cells 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 KD Validation of PHAPI in Human Breast Cancer Cells (Schafer et al., 2006) </strong><br> Human Breast Cancer Cells (T47D cells) were transfected with control or PHAPI siRNA duplex. PHAPI was detected via Western Blot analysis by using the anti-PHAPI antibody. PHAPI expression was reduced after PHAPI siRNA knockdown.
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<strong>Figure 7 Increased Expression Validation of PHAPI in Patient Samples of Breast Tumor Tissue (Schafer et al., 2006) </strong><br> PHAPI was overexpressed in all breast tumor samples of patients and human breast cancer cells (MDA-MB-453), but not in the normal breast tissue or human primary mammary epithelial cells (HMEC).
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<strong>Figure 8 Overexpression of PHAPI in Breast Cancer Cells (Schafer et al., 2006) </strong><br> Western blot analysis with anti-PHAPI antibodies was performed for PHAPI in human cell lines from breast, prostate and lung. PHAPI was overexpressed in breast cancer cells when compared with normal cells (HMEC) whereas there were no significant differences in PHAPI expression in normal and cancer cells of either prostate or lung origin.
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<strong>Figure 9 Induced Expression Validation of PHAPI/Anp32a in Atxn1 KO Mice (Sa?nchez et al., 2013) </strong><br> Western blot analysis of PHAPI/Anp32a from the cerebellum of WT and Atxn1 KO mice. PHAPI expression was significantly increased (2 folds) in Atxn1 KO mice as compared to WT mice. The same effect was observed in PHAPI mRNA levels.
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<strong>Figure 1 Western Blot Validation in Human Raji Cell Lysate</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PHAP I 3145 (A: 2 μg/mL, B: 4 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 Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PHAP I 3145 (2 μg/mL), PHAP I 3151 (1 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 Western Blot Validation in Human Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: PHAP I 3145 (2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Immunofluorescence Validation of PHAP I in Raji Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed Raji Cells labeling PHAP I with 3145 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
<strong>Figure 5 Immunocytochemistry Validation of PHAP I in Raji Cells</strong><br> Immunocytochemical analysis of Raji cells using anti-PHAP I antibody (3145) at 2 μg/ml. Cells 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 KD Validation of PHAPI in Human Breast Cancer Cells (Schafer et al., 2006) </strong><br> Human Breast Cancer Cells (T47D cells) were transfected with control or PHAPI siRNA duplex. PHAPI was detected via Western Blot analysis by using the anti-PHAPI antibody. PHAPI expression was reduced after PHAPI siRNA knockdown.
<strong>Figure 7 Increased Expression Validation of PHAPI in Patient Samples of Breast Tumor Tissue (Schafer et al., 2006) </strong><br> PHAPI was overexpressed in all breast tumor samples of patients and human breast cancer cells (MDA-MB-453), but not in the normal breast tissue or human primary mammary epithelial cells (HMEC).
<strong>Figure 8 Overexpression of PHAPI in Breast Cancer Cells (Schafer et al., 2006) </strong><br> Western blot analysis with anti-PHAPI antibodies was performed for PHAPI in human cell lines from breast, prostate and lung. PHAPI was overexpressed in breast cancer cells when compared with normal cells (HMEC) whereas there were no significant differences in PHAPI expression in normal and cancer cells of either prostate or lung origin.
<strong>Figure 9 Induced Expression Validation of PHAPI/Anp32a in Atxn1 KO Mice (Sa?nchez et al., 2013) </strong><br> Western blot analysis of PHAPI/Anp32a from the cerebellum of WT and Atxn1 KO mice. PHAPI expression was significantly increased (2 folds) in Atxn1 KO mice as compared to WT mice. The same effect was observed in PHAPI mRNA levels.

Documents

Further Information

Additional Names:
PHAP I Antibody: LANP, MAPM, PP32, HPPCn, PHAP1, PHAPI, I1PP2A, C15orf1, LANP, Acidic leucine-rich nuclear phosphoprotein 32 family member A, Acidic nuclear phosphoprotein pp32
Application Note:
WB: 2-4 μg/mL; ICC: 2 μg/mL; IF: 10 ?g/mL.

Antibody validated: Western Blot in human, mouse and rat samples; Immunocytochemistry in human samples; Immunofluorescence in human samples. All other applications and species not yet tested.
Background:
PHAP I Antibody: Apoptosis is related to many diseases and development. Caspase-9 plays a central role in cell death induced by a variety of apoptosis activators. Cytochrome c, after released from mitochondria, binds to Apaf-1, which forms an apoptosome that in turn binds to and activate procaspase-9. Activated caspase-9 cleaves and activates the effector caspases (caspase-3, -6 and -7), which are responsible for the proteolytic cleavage of many key proteins in apoptosis. The tumor suppressor putative HLA-DR-associated proteins (PHAPs) were recently identified as important regulators of mitochondrion apoptosis. PHAP appears to facilitate apoptosome-medicated caspase-9 activation and to stimulate the mitochondrial apoptotic pathway. PHAP was also shown to oppose both Ras- and Myc-medicated cell transformation.
Background References:
  • Jiang et al. Science. 2003;299(5604):223-6.
  • Nicholson and Thornberry. Science. 2003 10;299(5604):214-5.
Buffer:
PHAP I 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.
Immunogen:
Anti-PHAP I antibody (3145) was raised against a peptide corresponding to 15 amino acids near the carboxy terminus of human PHAP I.

The immunogen is located within the last 50 amino acids of PHAP I.
ISOFORMS:
Human PHAP I has one isoform (249aa, 28.6kD). Mouse PHAP I has one isoform (247aa, 28.5kD) and rat PHAP I also has one isoform (247aa, 28.6kD). 3151 can detect all isoforms of human, mouse and rat.
NCBI Gene ID #:
8125
NCBI Official Name:
acidic (leucine-rich) nuclear phosphoprotein 32 family, member A
NCBI Official Symbol:
ANP32A
NCBI Organism:
Homo sapiens
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
Predicted: 29kD

Observed: 29 kD
Protein Accession #:
P39687
Protein GI Number:
730318
Purification:
PHAP I Antibody is DEAE purified.
Research Area:
Apoptosis
SPECIFICITY:
This polyclonal antibody has no cross-reaction to PHAP I2a and PHAP III.
Swissprot #:
P39687
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

Independent Antibody Validation in Cell lines (Figure 2) shows similar PHAP I expression profile in human, mouse and rat cell lines detected by two independent anti-PHAP I antibodies that recognize different epitopes, 3145 against C-terminus domain and 3151 against C-terminus different domain. PHAP I proteins are detected in the most tested cell lines at different expression levels by the two independent antibodies.

KD validation (Figure 6): Anti-PHAP I antibody (3151) specificity was further verified by PHAP I specific knockdown. PHAP I signal in T47D cells transfected with PHAP I siRNAs was disrupted in comparison with that in cells transfected with control siRNAs.

Regulated expression validation (Figure 7, 8, 9): PHAP I expression detected by anit-PHAP I antibodies (3151) was up-regulated in breast cancer tissues of patients (Figure 7) and the breast cancer cell line (Figure 8) as well as in the Atxn1 KO mice (Figure 9).

References

  1. Schafer et al. Enhanced sensitivity to cytochrome c-induced apoptosis mediated by PHAPI in breast cancer cells. Cancer Res. 2006;66(4):2210-8. PMID: 16489023
  2. S?nchez et al. A novel function of Ataxin-1 in the modulation of PP2A activity is dysregulated in the spinocerebellar ataxia type 1. Hum Mol Genet. 2013 22(17):3425-37. PMID: 23630944
  3. Kurokawa et al. A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53. Sci Signal. 2013;6(274):ra32. PMID: 23652204
  4. Parrish. Regulation of Apoptosis Following Mitochondrial Cytochrome c Release. Department of Pharmacology and Molecular Cancer Biology, Duke University. PhD Thesis, 2010.

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