PA1-026A

antibody from Invitrogen Antibodies

Targeting: FKBP1A FKBP-12, FKBP1, FKBP12, FKBP12C, PKC12, PPIASE

Provider product page for PA1-026A

Western blot

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [41]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [3]
Other assay [14]

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Product number: PA1-026A - Provider product page
Provider: Invitrogen Antibodies
Product name: FKBP12 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-026A detects FK506 binding protein 12 kDa (FKBP12) in human and mouse tissues. PA1-026A has been successfully used in Western blot, immunohistochemistry, and immunoprecipitation procedures. By Western blot, this antibody detects a 12 kDa protein representing FKBP12 from rat heart homogenate. The PA1-026A immunogen is a synthetic peptide corresponding to the N-terminal residues G(1) V Q V E T I S P G D G R(13) of human FKBP12. PA1-026A immunizing peptide (Cat. # PEP-012) is available for use in neutralization and control experiments.
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

FKBP1A protein structure - PA1-026A shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized human Colon carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing FKBP12 (Product # PA1-026A) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized human Heart tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a rabbit polyclonal antibody recognizing FKBP12 (Product # PA1-026A) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized human Tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a rabbit polyclonal antibody recognizing FKBP12 (Product # PA1-026A) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 Cyclophilin A and FKBP12 are required for Tat palmitoylation and Tat-mediated inhibition of PI(4,5)P 2 -dependent membrane traffic. a PC12 cells were transfected with Tat-FLAG before treatment for 6 h with 1 uM FK506, 2 uM CSA, 1 uM rapamycin or solvent. Palmitoylation was then assayed using acyl-biotin exchange before band quantification. Results are expressed as biotin/Tat intensity ratio (% of control). b Jurkat T-cells were transfected with Tat before adding drugs for 6 h and assaying Tat secretion by ELISA. c PC12 cells were transfected with GH before drug treatment for 6 h and GH secretion assay. d Human MDMs were treated with the drug for 5 h before assaying phagocytosis of IgG-coated beads. e PC12 cells were transfected with the indicated siRNA before lysis then anti-CypA or anti-FKBP12 then anti-alphatubulin western blot. f PC12 cells were cotransfected with the indicated siRNA and Tat-FLAG before detecting Tat palmitoylation using acyl-biotin exchange. (HA, hydroxylamine). g Jurkat cells were cotransfected with Tat and the indicated siRNA (silencing efficiency is shown in Supplementary Fig. 12b ) and Tat secretion was assayed after 48 h. Data are mean +- SEM, n = 3 independent experiments. The significance of differences with controls was assessed using ANOVA, one-way ( a , b , g ) or two-way ( c , d ) (*** p < 0.001; ** p < 0.01; * p < 0.05)

Supportive validation

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Experimental details: Figure 4 Aging does not change significantly the expression of FKBP12 protein ( A ) Representative immunoblotts and ( B ) average +- sem values (arbitrary units) (n=6) of the FKBP12 expression in muscular layer of colon from young and aged guinea pigs.

Supportive validation

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Experimental details: Figure 2 Bone morphogenetic protein (BMP) signaling components in the Muller glia of adult mouse retina. A : Sections of 4 week-old retina were subjected to double-label immunofluorescence with antibodies specific for sex determining region Y box 2 (SOX2), which labels Muller glia, retinal astrocytes, and cholinergic amacrine cells, and intracellular members of the BMP pathway SMAD1 . SMAD1 (+) cells were localized to the inner nuclear layer (INL) and ganglion cell layer (GCL) of P30 retina. A subpopulation of SOX2 (+) Muller glia (arrows) and retinal astrocyte cells (arrowheads) were also positive for SMAD1. B : Sections of 4 week-old retina were subjected to double-label immunofluorescence with antibodies for SOX2, and TGF-beta activated kinase 1 (TAK1). Cells positive for the protein TAK1 were found to be localized in the INL and GCL. C : Sections of 4 week-old retina were subjected to double-label immunofluorescence with antibodies for SOX2, and FK506 rapamycin binding protein (FKBP12). FKBP12, also part of the non-canonical BMP pathway, was found to be localized in the INL along with sparse localization in the GCL. Very little to no co-label was seen with glutamine synthetase (GS; +) and TAK1 or FKBP12 (+) cells ( B , C ). n=3 different eyes for each label. Scale Bar A=50 mum applies to all panels.

Supportive validation

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Experimental details: Figure 2. Protein content in patients' microsomes. ( A ) Western blot analysis of glycogen phosphorylase (GP) for 25 subjects, in a luminescence scale with black as 0 and white as saturating value. The numbers above each lane are patient identifiers that apply to all 25-lane gels shown in the article. The twelve lanes under the black bar have protein from MHN patients; those under the red bar are from MHS patients, except #144, which was reclassified as MHN. ( B ) Ponceau-stained gel that originated all blots in the figure. A and B illustrate our custom quantitative analysis. The content of every protein quantified in blots was calculated as the signal mass within region a above background (average level in b). Content was normalized for quantity of preparation in the lane dividing by the signal similarly calculated in the gel in B. The signal in B is computed in a large area of the lane to average multiple proteins in the fraction. The method is fully demonstrated in Video 1 . Arrows in B mark two bands, near 100 and 180 kDa, with a visibly greater signal in the MHS group. Their main components were GP and glycogen debranching enzyme (GDE) respectively. ( C-E ) Western blots of GDE, calsequestrin 1 and FKBP12 in different sections of gel B (identified by molecular weight markers at left). ( F and G ) Box plots of GP and GDE content. In both cases the content was greater on average for MHS and the differences significant (data in Table 1 ). ( H ) GP vs. GDE in blots A and C;

Supportive validation

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Experimental details: Fig 1 Characterization of stably transfected Daoy cell lines that allow for pharmacological controlled activation of PERK. (A) Daoy cells were transfected with the plasmid pIREs-Fv2E-PERK-ZsGreen. We obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK. (B) Western blot analysis showed that AP20187 treatment activated Fv2E-PERK and led to phosphorylation of eIF2alpha. The positions of activated Fv2E-PERK (p-Fv2E-PERK) and inactive Fv2E-PERK proteins were indicated. (C) Densitometry analysis of western blot results showed that AP20187 treatment increased the level of p-eIF2a in Fv2E-PERK1 cells in a dose-dependent manner. The relative protein levels are relative to actin. (D) SUnSET measurement of protein biosynthesis revealed dramatic reduction of protein biosynthesis in Fv2E-PERK1 cells treated with the high dose of AP20187 (0.1-1 nM). Nevertheless, treatment with the low dose of AP20187 (0.001-0.01 nM) only slightly reduced protein biosynthesis in the cells. (E) MTT analysis showed that treatment with the high dose of AP20187 (0. 1-1 nM) significantly inhibited Fv2E-PERK1 cell growth. Nevertheless, treatment with the low dose of AP20187 (0.001-0.01 nM) had no effect on the cell growth. The experiments were repeated at least three times. Error bars represent SD, * P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 10.1371/journal.ppat.1009022.g002 Fig 2 Inhibition of calcineurin phosphatase activity enhances VZV gB/gH-gL mediated cell fusion. (A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean +- standard error of the mean (SEM) for >=3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 mum. (F) Tacrol

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 10.1371/journal.ppat.1009022.g003 Fig 3 Pimecrolimus disruption of calcineurin-dependent regulation of gB/gH-gL fusion requires FKBP1A. (A) FKBP1A shRNA knockdown (KD) in MeWo cells. FKBP1A Transcript levels detected by RT-qPCR in MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells were normalized to housekeeping gene PGM1 and presented as a percentage of control cells, with mean +- SEM from three independent experiment (two-tailed, unpaired t-test, ****, p < 0.0001). Western blot of FKBP1A protein (anti-FKBP1A). FKBP1A band density was normalized to alpha-tubulin in control and FKBP1A KD cells respectively. The ratio of the two values was presented as a percentage of control cells. A representative picture, and mean +- SEM of the ratio from three independent experiments are shown. (B) Quantification of calcineurin phosphatase activity in FKBP1A KD cells using the NFATC1 nuclear translocation assay. MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells transiently expressing GFP-NFATC1 were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 muM) 30 min before ionomycin (Iono, 1 muM; 30 min). Fluorescence microscopy images of GFP-NFATC1 (green), nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 mum. Red arrowheads indicate nucleus localization ( N ), white arrowhead indicates diffused localization in both nucleus and cytoplasm ( N/C ), and white arrows indicate cytoplasm localization ( C ). Box and whisker plots of GFP-NFATC1 positi

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Drug targets. ( A ) Pie charts of primary targets of all approved drugs ( left ) and all phase I ( center ) and phase II ( right ) drug metabolism genes expressed in the intestinal epithelium shown by lineage with highest expression. ( B ) Primary targets of drugs used to treat IBD found to have expression in the intestinal epithelium. ( C ) Dotplots showing expression of primary targets of drugs used to treat IBD by lineage split into high-, middle-, and low-expressing tables for better visualization. Note scaling changes between tables. ( D ) Left : In situ hybridization showing BEST4 RNA (white) and immunofluorescence staining for FKBP1A protein (magenta) and nuclei (blue) in human jejunum ( top ) and colon ( bottom ). Center : Immunofluorescence staining for IMPDH2 protein (magenta), KI67 (white), and nuclei (blue) in human jejunum ( top ) and colon ( bottom ). Right : Immunofluorescence staining for DHFR protein (magenta), KI67 (white), and nuclei (blue) in human jejunum ( top ) and colon ( bottom ). Optical slice was 2 mum for all. Scale bars : 50 mum. ( E ) Dotplot showing expression of the top 3 highest-expressed targets of IBD drugs in the intestinal epithelium across regions and split by donor. FKBP1A, FKBP Prolyl Isomerase 1A; IMPDH2, Inosine Monophosphate Dehydrogenase 2; JAK, Janus Kinase; Sec. Prog., Secretory Progenitor.