PA1-901

antibody from Invitrogen Antibodies

Targeting: ITPR1 ACV, Insp3r1, IP3R1, PPP1R94, SCA15, SCA16, SCA29

Provider product page for PA1-901

Western blot

Antibody data

Antibody Data
Antigen structure
References [42]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [1]
Flow cytometry [1]
Other assay [22]

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Product number: PA1-901 - Provider product page
Provider: Invitrogen Antibodies
Product name: IP3 Receptor 1 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-901 detects inositol 1,4,5-trisphosphate receptor type-I (IP3R-I) from canine, mouse and rat tissues.
Concentration: 1.3 mg/mL

ITPR1 protein structure - PA1-901 shown in red.

Supportive validation

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Experimental details: Figure 1 Identification and validation of the anti-inositol 1,4,5-triphosphate receptor 1 (IP3R1) antibody Patient sera with a working diagnosis of cerebellar ataxia were screened using immunohistochemistry in mouse brain slices. A specific pattern was identified, where the antibodies bound primarily the Purkinje cells (A). Following antibody identification, the precipitate used for the mass spectrometry was blotted and probed with a commercial antibody against IP3R1. The antibody recognized the band, with an apparent molecular weight of 270-280 kDa (lane 1). The same antibody did not recognize precipitates from healthy control subjects (lanes 2 and 3) (B). We double-labeled mouse brain with patient serum (C) and the commercial antibody against IP3R1 (D). The colocalization was near perfect, confirming the identity of the autoantibody (E). (A) Scale bar 200 mum; (C-E) scale bar 100 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Experimental induction ER-mitochondria interactions increases mitochondrial calcium flux and impairs glucose homeostasis (A) Schematic illustrating the mitochondrial-ER synthetic linker. The construct encodes a monomeric red fluorescent protein (RFP) fused to the OMM targeting sequence of mAKAP1 at the N terminus and the ER targeting sequence of yUBC6 at the C terminus with a length of 25 nm 25 . Representative TEM of Hepa 1-6 cells expressing control (upper panel) and linker plasmids (bottom panel) at 9300x, scale bar: 500nm. (B) FACS analysis of Annexin V positivity in control and linker-expressing cells, n=3. (C) Representative trace of mitochondrial Ca 2+ dynamics in single Hepa 1-6 cells expressing the control or linker construct, measured by FRET of 4mtD3cpv following treatment with ATP (100muM). (C inset) Quantification of the peak in Ca 2+ after ATP stimulation, 3 independent experiments. (D) Measurement of oxygen consumption rate (OCR) in control and linker-expressing Hepa 1-6 cells in the presence of 2muM Oligomycin, 0.4 muM FCCP and 2muM Rotenone (Rot) and 2muM antimycin (AA). n=15 from 4 independent experiments. (E) Representative TEMs of liver sections from lean mice expressing the control or linker construct, scale bar: 500nm upper panel and 100nm bottom panel (F) Liver sections from HFD-fed mice expressing control or linker constructs, stained with hematoxylin and eosin, scale bar 50um. (G) OCR of primary hepatocytes from HFD-fed mice expressing contro

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Identification and validation of the anti-inositol 1,4,5-triphosphate receptor 1 (IP3R1) antibody Patient sera with a working diagnosis of cerebellar ataxia were screened using immunohistochemistry in mouse brain slices. A specific pattern was identified, where the antibodies bound primarily the Purkinje cells (A). Following antibody identification, the precipitate used for the mass spectrometry was blotted and probed with a commercial antibody against IP3R1. The antibody recognized the band, with an apparent molecular weight of 270-280 kDa (lane 1). The same antibody did not recognize precipitates from healthy control subjects (lanes 2 and 3) (B). We double-labeled mouse brain with patient serum (C) and the commercial antibody against IP3R1 (D). The colocalization was near perfect, confirming the identity of the autoantibody (E). (A) Scale bar 200 mum; (C-E) scale bar 100 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Determination and immunolocalization of the expression pattern of IP3R proteins. (A) Western blot analysis of IP3R-1 (a), IP3R-2 (b), and IP3R-3 (c), respectively, in SolC1(-) and SolD(+) myotubes. 30 mug protein from each type of myotube was separated on 5% SDS-PAGE, transferred onto a pore nitrocellulose membrane, and processed for Western blot analysis with specific antibodies. The 220-kD band corresponded to the molecular weight of the myosin protein. (B) IP3R isoform immunolabeling in SolC1(-) and SolD(+) myotubes was observed with laser scanning confocal microscopy. IP3R-1 and IP3R-2 localizations were detected using polyclonal antibodies, PA1-901 and PA1-904, respectively. IP3R-1 staining is localized in the nuclear envelope region and in the cytoplasm in SolC1(-) (a) and in SolD(+) (c) myotubes. IP3R-2 showed a peripheral and fully cytoplasmic localization and, as indicated with white arrows, striated staining pattern appeared at this differentiation stage in SolC1(-) (b) and SolD(+) (d) myotubes. Bars, 25 mum.

Supportive validation

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Experimental details: Figure 7. Immunolocalization of RyRs and IP3Rs in SolC1(-) and SolD(+) myotubes. Immunolabelings of SR calcium release channels in SolC1(-) (A-C) and in SolD(+) myotubes (D-F) were observed with laser scanning confocal microscopy (LSCM). The same random distribution of RyRs (anti-RyR; Calbiochem) was observed in SolC1(-) myotubes (A) and in SolD(+) myotubes (D). In the same myotubes, immunostaining with antibodies against IP3R-2 was performed in SolC1(-) (B) and in SolD(+) (E). Yellow arrows, locations corresponding to elevated staining for IP3R-2 and low staining for RyR. IP3R-1 staining is localized in the nuclear envelope region and in the cytoplasm in SolC1(-) (C) and in SolD(+) (F) myotubes. Insets, staining of selected nuclei (dotted squares) loaded with the fluorescent TO-PRO-3 probe. IP3R-1 and IP3R-2 localizations were detected using polyclonal antibodies, PA1-901 and PA1-904, respectively.

Supportive validation

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Experimental details: Figure 3 Regulation of MAM enriched proteins in obesity (A) Schematic illustration of functional and structural MAM proteins. (B) Western blot and image J based quantification analysis of indicated proteins in liver total lysates from wt/ ob/ob and lean/HFD-fed (16 weeks) mice. Arrow indicates the specific Sig1R band, n=3 representative of 2-3 independent experiments. (C) Western blot analysis of indicated proteins of subcellular fractions from mouse livers. CM: crude mitochondria. PM: Pure mitochondria, ER: endoplasmic reticulum and MAM: mitochondria associated ER membranes. Cytochrome C (Cyto C) was used as a mitochondrial marker; IP3R and SERCA were found mostly in the bulk ER. PDI and Calnexin were equally distributed between ER and MAM, and Sigma 1 receptor (Sig1R) enrichment served as a known MAM marker. This figure is representative of at least 5 different preparations (D) Western blot and quantification analysis of indicated proteins in MAMs from wt/ ob/ob and lean/HFD mice, n=3. PDI served as a loading control. All the graphs represent mean +- SEM, *P < 0.05, Student's t-test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Experimental suppression of MAM function or formation alters mitochondrial calcium flux and improves glucose homeostasis (A) Western blot and quantification analysis demonstrating specific knockdown of IP3R1 in liver lysates following adenoviral shRNA transduction, n= 3, representative of 2 independent experiments. (B) Oxygen consumption rate of primary hepatocytes from mice expressing control or shIP3R1, n=6. (C) Western blot and quantification analysis showing reduced JNK phosphorylation in liver lysates following IP3R1 knockdown. (D) Western blot and quantification analysis of the insulin-action in the liver samples following expression of shScramble or shIP3R1. n=3. (E) Glucose tolerance test in mice expressing shScramble or shIP3R1, n=7. (F) Western blot and quantification analysis demonstrating specific knockdown of PACS-2 in liver lysates following adenoviral shRNA transduction, n=4 representative of 2 independent experiments. (G) Oxygen consumption rate of primary hepatocytes from mice expressing control or shPACS-2, n=5 for shscramble and n=8 for shPACS-2. (H) Western blot and quantification analysis of the insulin-action in the liver samples following expression of shScramble or shPACS-2, n=3. (I) Glucose tolerance test in mice expressing shScramble or shPACS-2, n=7 for shScramble and 9 for shPACS-2. (J) We propose here that an early development in the course of obesity-induced metabolic disease is increased MAM formation in the liver. Increased MAM formati

Supportive validation

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Experimental details: Fig. 6 A specific therapeutic peptide blocks the FUNDC1-IP3R1 interaction and angiogenesis. a Transduction efficiency of peptides 1, 2, and a control peptide in Human umbilical vein endothelial cells (HUVECs) ( n = 5 independent experiments). b HUVECs were treated with different peptides (20 muM) for 24 h, then subjected to immunoprecipitation with antibody against FUNDC1(HA) to quantify the interaction of FUNDC1 and IP3R1 ( n = 3 independent experiments). c HUVECs were treated with different peptides (20 muM) for 24 h, then cytosolic Ca 2+ was detected using the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). d HUVECs were treated with different peptides. After 24 h, the cell lysates were collected and subjected to western blot assays to detect the expression of VEGFR2, IP3R1, SRF, and phosphorylated SRF at Ser103 (pSRF) ( n = 5 independent experiments). e Three-dimensional spheroids and representative images of spheroid-sprouting were analyzed ( n = 5 independent experiments). Scale bar, 100 um. f Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into 6-week-old wild-type mice, then the mice received an intravenous injection of the indicated peptides. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assay ( n = 5 mice/group). Quantification of Hb extracted from matrigel plugs of different groups. g C57BL/6 mice with LLC tumors that were approximately 90 mm 3 in size

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Increased expression of endoplasmic reticulum (ER) stress sensor proteins and ER-mitochondrial axis proteins due to stigmasterol treatment in ovarian cancer cells. ( A , B ) Immunoblots representing the expression of unfolded protein response (UPR) proteins following stigmasterol treatments (0, 5, 10, and 20 mug/mL). TUBA was used as a control and is shown at the bottom of the figure. ( C , D ) Immunoblots representing the expression of ER-mitochondrial axis proteins following stigmasterol treatments (0, 5, 10, and 20 mug/mL). TUBA was used as a control and is shown at the bottom of the figure. ( E , F ) Immunoblots representing the expression of autophagy proteins following stigmasterol treatment. TUBA was used as a control and is shown at the bottom of the figure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 beta2-AR agonists enhance physical contacts between ER and Mito. ( A ) Representative images of PLA in HeLa cells treated with DMSO or isoproterenol (1 muM). PLA signal (red) indicates a close contact between ER and Mito. Sec61-GFP (green): to label ER; DAPI (blue): nucleus. ( B ) Quantifications of PLA signal as shown in A ( n = 4 experiments with 77-97 cells each; Mann-Whitney test). ( C ) Representative TEM images of Mito (HEK293T cells) showing no contact (left) or a close contact (right) with ER membrane. Red lines: the distance between ER and Mito membrane. ( D ) Quantitative analysis of the percentage of the number of Mito showing contact or no contact with ER ( n = 3 experiments with 346, 405 Mito for control, isoprot; Chi-square test). ( E , F ) The percent of total contact length divided by the total Mito perimeter measured among all mitochondria ( E ) ( n = 4 experiments with 122, 171 mitochondria for control, isoprot; Mann Whitney test), and the percent of contact length divided by Mito perimeter using only Mito showing a close contact with ER ( F ) ( n = 4 experiments with 27, 95 mitochondria for control, isoprot; Mann Whitney test) in TEM images. ( G ) Representative images of Western blots of biotinylated proteins proximity-labeled by Mito-APEX (for 3 min) followed by streptavidin affinity pull down, in control- vs isoprot-treated HEK293T cells. GAPDH used for control. Full-length blots are presented in Supplementary Fig. S6 . ( H ) Quantification of I

Supportive validation

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Experimental details: Fig. 4 iRhoms bind to IP 3 Rs. a Levels of iRhom2 and IP 3 R1 were analysed by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA or IP: FLAG) from HEK293T cells transiently transfected for 36 h with FLAG-IP 3 R1 and iRhom2-HA. b Levels of iRhom2 and endogenous IP 3 R1, STIM1 and SERCA2 were determined by immunoblotting in whole cell lysate (WCL) and after immunoprecipitation (IP: HA) from iRhom1/2 DKO MEFs stably expressing iRhom2-HA. c Proximity ligation assay (PLA) in iRhom1/2 DKO HEK293T cells reconstituted with HA-iRhom2 under a tetracycline-inducible promoter were stained using antibodies to detect endogenous IP 3 R1 and N-terminally HA-tagged iRhom2 after 24 h of 250 ng/ml of doxycycline to induce iRhom2. Dots per cell were quantified using ImageJ and shown as mean +- SEM, uninduced ( n = 4 images/166 cells), induced ( n = 7 images/259 cells) from two biologically independent experiments. Two-tailed unpaired Student's t -test **** p < 0.0001. Bottom panels are magnified sections of above corresponding panels. Scale bars = 10 mum. d Levels of WT and deletion mutants of iRhom2 and endogenous IP 3 R1 were determined by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from iRhom1/2 DKO MEFs stably expressing iRhom2-WT-HA or iRhom2-DeltaIRHD-HA or iRhom2-DeltaN-HA. Schematics show iRhom2 mutants with domains deleted (numbering refers to TMDs and IRHD refers to iRhom homology domain). e Levels of WT and deletion mutant of iRho

Supportive validation

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Experimental details: Fig. 6 iRhoms control BCL-2 antiapoptotic effect on IP 3 Rs. a Cell lysates from WT and iRhom1/2 DKO MEFs treated with tunicamycin (0.5 ug/ml) alone or in the presence of BIRD-2 (10 uM) or ABT-199/Venetoclax (ABT, 1 uM) were analysed by immunoblotting with indicated antibodies. b Levels of iRhom2 and endogenous IP 3 R1, IP 3 R3 and BCL-2 were analysed by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: HA) from iRhom1/2 DKO MEFs stably expressing iRhom2-HA after treatment for 18 h with control or BIRD-2 peptides (10 uM or 20 uM). c , d Levels of iRhom2, IP 3 R1 and BCL-2 were analysed by immunoblotting in whole cell lysate (WCL) and immunoprecipitation (IP: FLAG) from HEK293T cells transiently transfected for 36 h with FLAG-IP 3 R1 (1.25 ug), iRhom2-HA (0.25 ug) and VENUS-BCL-2_ER (0.25 ug; BCL-2 targeted specifically to ER) in ( c ) or GFP-IP 3 R1 (1.25 ug), iRhom2-HA (0.25 ug) and FLAG-BCL-2_ER (0.25 ug; Bcl-2 targeted specifically to ER) in ( d ). Immunoblotting data are representative of 2-3 independent experiments. e Proposed model for the role of iRhoms in regulating IP 3 R function via BCL-2. In WT cells, IP 3 R, iRhoms and BCL-2 interact at the ER within a regulatory complex, where iRhom2 attenuates the inhibitory effect of BCL-2 on IP 3 R activity. This enhances the flow of Ca 2+ from the ER into mitochondria under basal conditions and during acute release of Ca 2+ (e.g. ER stress) to regulate apoptosis. In the absence of iRhoms, the inhibitory

Supportive validation

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Experimental details: Alpinumisoflavone activates the ER stress and ER-mitochondria contact proteins in ES2 and OV90 cells. ( A , B ) In both ES2 ( A ) and OV90 ( B ) cells treated with alpinumisoflavone for 24 h, immunoblots of GRP78, phosphor-eIF2alpha, total-eIF2alpha, VDAC, IP3R1 and TUBA were captured using the ChemiDoc system. Relative intensities of each target protein are presented as bar graphs next to sorted immunoblots. All experiments were performed in triplicate. The significance between the vehicle and treatment groups is depicted by asterisks (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Supportive validation

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Experimental details: Typical outcome of Purkinje cell imaging in 3D reconstruction with a single antibody (A and B) 3D image reconstruction of Purkinje neurons acquired at 20x ( left ) and 63x ( right ) in wild-type mice at 10 weeks of age with staining against Calbindin (A, red) or IP3R1 (B, green).

Supportive validation

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Experimental details: Figure 1 Relative levels of CRU proteins in crude homogenates from skeletal muscle. Identical amounts (25 ug/lane) of microsomal fraction of skeletal muscles from WT, Tdn-, Jct- and Tdn-/Jct mice were loaded and immunoblotted with several antibodies. Membranes were tested for expression of triadin (Tdn), junctin (Jct), ryanodine receptor (RyR1), Dihydropyridine receptors (Cav1.1), Calsequestrin (CASQ), SERCA-1 pump (SERCA), FK506 binding protein (FKBP12), Junctophilin-1 (JP-1) and Histidin-rich Ca 2+ binding protein (HRC) as described in Material and Methods. Band intensity for each protein was normalized to GAPDH expression to correct for loading and plotted as fraction of its WT counterpart (dotted line). Data presented as mean +- SD of 3-7 independent blots. * p

Supportive validation

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Experimental details: 8 Figure Osthole influenced intracellular mechanisms related to endoplasmic reticulum (ER)-mitochondrial functions. Expression of ER-mitochondrial function targets such as (a) ULK1, (b) GRP75, (c) MFN2, (d) P62, (e) FAM82A2, (f) VAPB, (g) VDAC, (h) IP3R1, and (i) IP3R2 was analyzed by western blot analysis in ES2 and OV90 cells treated with various doses of osthole (0, 5, 10, and 20 muM). Intensity of the target protein was detected and analyzed relative to total protein or alpha-tubulin protein. *** p < .001, ** p < .01, and * p < .05 indicate significant differences compared to vehicle-treated cells. IP3R1, inositol 1,4,5-trisphosphate (IP3) receptor type 1; IP3, inositol trisphosphate; MFN2, mitofusin-2; VAPB, vesicle-associated membrane protein-associated protein B/C; VDAC, voltage-dependent anion channel

Supportive validation

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Experimental details: Figure 6 Stimulation of endoplasmic reticulum (ER) stress sensor, ER-mitochondria tethering proteins, and autophagy regulators by fucoidan in ES-2 and OV-90 cells. ( A - F ) The stimulation of ER stress sensor proteins, ( A ) IRE1alpha, ( B ) ATF6alpha, ( C ) p-PERK, ( D ) GADD153, ( E ) p-eIF2alpha, and ( F ) GRP78 was observed through western blot analysis in ES-2 and OV-90 cell lines incubated with fucoidan. ( G - L ) The activation of ER-mitochondria axis-associated proteins, ( G ) VDAC, ( H ) IP3R1, ( I ) IP3R2, ( J ) GRP75, ( K ) MFN2, and ( L ) VAPB was analyzed by western blotting in fucoidan treated ES-2 and OV-90 cells. ( M - O ) The activation of autophagy-associated proteins, ( M ) LC3B, ( N ) BECN1, and ( O ) ATG5 was observed through western blot in ES-2 and OV-90 cells incubated with fucoidan. The graph of the signals was calculated compared with total signal or alpha tubulin (TUBA). *** p < 0.001, ** p < 0.01, and * p < 0.05 show significances.