32-5000
antibody from Invitrogen Antibodies
Targeting: PTBP1
HNRNP-I, HNRPI, pPTB, PTB, PTB-1, PTB2, PTB3, PTB4
Antibody data
- Antibody Data
- Antigen structure
- References [6]
- Comments [0]
- Validations
- Western blot [2]
- Immunocytochemistry [1]
- Other assay [9]
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- Product number
- 32-5000 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- PTBP1 Monoclonal Antibody (7)
- Antibody type
- Monoclonal
- Antigen
- Recombinant full-length protein
- Reactivity
- Human
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- 7
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- -20°C
Submitted references The RNA-Binding Proteins SRP14 and HMGB3 Control HIV-1 Tat mRNA Processing and Translation During HIV-1 Latency.
HIV latency reversing agents act through Tat post translational modifications.
Interleukin 7 up-regulates CD95 protein on CD4+ T cells by affecting mRNA alternative splicing: priming for a synergistic effect on HIV-1 reservoir maintenance.
Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.
Gestational methylazoxymethanol exposure leads to NMDAR dysfunction in hippocampus during early development and lasting deficits in learning.
The polypyrimidine tract-binding protein (PTB) is involved in the post-transcriptional regulation of human inducible nitric oxide synthase expression.
Khoury G, Lee MY, Ramarathinam SH, McMahon J, Purcell AW, Sonza S, Lewin SR, Purcell DFJ
Frontiers in genetics 2021;12:680725
Frontiers in genetics 2021;12:680725
HIV latency reversing agents act through Tat post translational modifications.
Khoury G, Mota TM, Li S, Tumpach C, Lee MY, Jacobson J, Harty L, Anderson JL, Lewin SR, Purcell DFJ
Retrovirology 2018 May 11;15(1):36
Retrovirology 2018 May 11;15(1):36
Interleukin 7 up-regulates CD95 protein on CD4+ T cells by affecting mRNA alternative splicing: priming for a synergistic effect on HIV-1 reservoir maintenance.
Yin Y, Zhang S, Luo H, Zhang X, Geng G, Li J, Guo X, Cai W, Li L, Liu C, Zhang H
The Journal of biological chemistry 2015 Jan 2;290(1):35-45
The Journal of biological chemistry 2015 Jan 2;290(1):35-45
Identification of cis-acting elements and splicing factors involved in the regulation of BIM Pre-mRNA splicing.
Juan WC, Roca X, Ong ST
PloS one 2014;9(4):e95210
PloS one 2014;9(4):e95210
Gestational methylazoxymethanol exposure leads to NMDAR dysfunction in hippocampus during early development and lasting deficits in learning.
Snyder MA, Adelman AE, Gao WJ
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2013 Jan;38(2):328-40
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2013 Jan;38(2):328-40
The polypyrimidine tract-binding protein (PTB) is involved in the post-transcriptional regulation of human inducible nitric oxide synthase expression.
Pautz A, Linker K, Hubrich T, Korhonen R, Altenhöfer S, Kleinert H
The Journal of biological chemistry 2006 Oct 27;281(43):32294-302
The Journal of biological chemistry 2006 Oct 27;281(43):32294-302
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Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis was performed on nuclear enriched extracts of A549 (Lane 1), HT-29 (Lane 2), HEK-293 (Lane 3), HeLa (Lane 4) and Hep G2 (lane 5). The blot was probed with Anti-PTBP1 Mouse Monoclonal Antibody (Product # 32-5000, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 57 kDa band corresponding to PTBP1 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).
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- Knockdown of PTBP1 was achieved by transfecting HeLa cells with PTBP1 specific validated siRNAs (Silencer® select Product # s11434). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the PTBP1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with PTBP1 Monoclonal Antibody (Product # 32-5000, 2 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PTBP1 .
Supportive validation
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- Immunofluorescence analysis of PTB (Clone 7) was performed using 90% confluent log phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with PTBP1 (7) Mouse Monoclonal Antibody (Product # 32-5000) at 2µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
Supportive validation
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- Figure 3 PTBP1 represses inclusion of endogenous BIM exon 3. (A) +2,582 to +2,662 of the polymorphic fragment has been expanded to show the nucleotide sequence. The three predicted PTBP1 binding sites are boxed. (B) Western blot analysis for PTBP1 protein levels after nucleofecting K562 cells with three different PTBP1-specific siRNA duplexes. Equal loading in each lane was determined by blotting of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ""U"" represents control cells that were not subjected to nucleofection. (C) Real-time RT-PCR analysis of RNA from K562 cells nucleofected with either control or PTBP1-specific siRNA duplexes to determine the ratio of endogenous exon 3- to exon 4-containing BIM transcripts. ""U"" represents control cells that were not subjected to nucleofection. Results are presented as an average of three biological replicates and the relative endogenous BIM E3: E4 ratio was determined by normalizing to the E3: E4 ratio of K562 cells that were not nucleofected. Error bars represent +- SEM. ** p
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- Figure 7 Knockdown of hnRNP H and hnRNP F does not promote inclusion of BIM exon 3. K562 cells were nucleofected with two different siRNA duplexes targeting either hnRNP H (siH1, siH2) or hnRNP F (siF1, siF2). Some cells were also nucleofected with siRNA duplexes targeting both hnRNP H and F (siF1 and siH1, siF2 and siH2). 48 hours later, real-time RT-PCR analysis of RNA from K562 cells was performed to determine the relative expression of (A) hnRNP H and (B) hnRNP F transcripts. ""U"" represents control cells that were not subjected to nucleofection. Results are presented as an average of three biological replicates and data is shown as mean +- SEM relative to non-nucleofected cells. (C) Western blot showing the efficacy of the two siRNA duplexes targeting hnRNP H. Protein bands shown are from different lanes but within the same membrane. Equal loading in each lane was determined by blotting of beta-actin. (D) Ratio of endogenous BIM transcripts harboring exon 3 over exon 4 after nucleofecting K562 cells with the indicated siRNAs. ""U"" represents control cells that were not subjected to nucleofection. Results are presented as an average of three biological replicates and the relative endogenous E3: E4 ratio was determined by normalizing to the endogenous E3: E4 ratio of K562 cells that were not nucleofected with siRNA. Error bars represent +- SEM.
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- Figure 9 SiRNA-mediated knockdown of PTBP1, but not hnRNP C, inhibits imatinib-induced apoptosis in CML cells. (A) K562 cells were nucleofected with either control or PTBP1-specific siRNA duplexes. 24 hours later, these cells were treated with increasing doses of imatinib (0, 0.25, 0.5 uM). The cells were harvested after another 24 hours and western blot was performed to determine the protein levels of PTBP1, PARP, cleaved caspase 3 and BIM. Equal loading in each lane was determined by blotting of GAPDH. (B) The same experiment was performed as described in (A). The only difference was that siRNA duplexes against hnRNP C were used instead of PTBP1-specific siRNA duplexes.
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- FIGURE 7 SRP14 and PTB expression induce HIV expression in primary CD4 + T-cells from patients on ART. (A) SRP14, HMGB3 and PTB protein expression in resting CD4 + T-cells isolated from patient under antiretroviral therapy (LK, leukophoresis) increase in response to T-cell stimulation by alpha-CD3/CD28 (1 mug/mL; 0.5 mug/mL). Expression of SRP14, HMGB3, and PTB was measured by western-blot in resting and activated CD4 + T-cells 72 h post-stimulation. GAPDH was used as a loading control and Jurkat protein lysate as a positive control for antibody detection of the various proteins. (B) SRP14, HMGB3, and PTB RNA levels were assessed by RT-qPCR and shown as fold change in activated (aCD4 + ) vs. resting CD4 + (rCD4 + ) after normalization on 18S RNA. (C) Effect of SRP14, HMGB3 and PTB overexpression following doxycycline (+Dox) treatment on HIV-1 RNA levels in the culture supernatant 48 h post-stimulation. (D) Plots depicting the expression of middle (CD25) and late (HLA-DR) activation markers on SRP14 transfected cells and PHA stimulated cells.