PA3-022

antibody from Invitrogen Antibodies

Targeting: PPID CYP-40

Provider product page for PA3-022

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Gel shift

Other assay

Antibody data

Antibody Data
Antigen structure
References [43]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [2]
Immunohistochemistry [2]
Other assay [26]

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Product number: PA3-022 - Provider product page
Provider: Invitrogen Antibodies
Product name: Cyclophilin 40 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

PPID protein structure - PA3-022 shown in red.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Skeletalmuscle 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 1:100 with a rabbit polyclonal antibody recognizing Cyclophilin D (Product # PA3-022) 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: Immunohistochemistry was performed on cancer biopsies of deparaffinized human Hepatocarcinoma 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 Cyclophilin D (Product # PA3-022) 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.

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Experimental details: Figure 6 Enlarged lipid droplets correlate with inhibition of HCV replication using siRNA knockdown . A) sg-1b replicon cells were transiently transfected with siRNA to target cypA, cypB and cyp40. A RISC-free siRNA construct was used as a control. Whole cell lysates were analyzed for target protein expression by Western blot at 72h and 96h post-transfection. B) Total RNA from siRNA transfected cells was extracted and used for quantitative real-time RT-PCR to measure copies of HCV. Replication in RISC-free control cells was set to 100% for each time point. Results are the average of 4 independent experiments, +- standard deviation. CypA and cyp40 siRNA result in a statistically significant decrease in replicon activity, p < 0.01 for cypA and p < 0.05 for cyp40 compared to RISC-free siRNA by Students paired t-test. C) At 96h post-transfection, lipid droplets were visualized as described. Representative images for each siRNA are shown. D) 20X images were acquired and quantified by the Cellomics ArrayScan VTI HCS reader. The mean fluorescence intensities for BODIPY 493/503 were normalized to the RISC free siRNA control. Those siRNA constructs that resulted in inhibition of HCV [ 17 ] also caused the lipid droplets to become enlarged, similar to what is seen with NIM811 treatment.

Supportive validation

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Experimental details: Figure 2 Over-expression of Cyp40, FKBP51 PCa cell lines. ( a ) WCEs from normal prostate epithelial cells (PrEC), androgen-dependent (AD) (LNCaP, LAPC-4, VCaP and DuCaP) and- independent (AI) (C4-2, C-81 and CWR22R) human PCa cell lines were prepared and analyzed by Western blot for expression of the AR, FKBP51, Cyp40 and actin, respectively, as described in Materials and Methods. ( b ) Densitometric scanning of the bands corresponding to protein expression of Cyp40, FKBP51 and the AR in PrEC, AD and AI human prostate cell lines. Cyp40, FKBP51 and AR protein levels were expressed as relative expression levels, taking the values obtained from LNCaP cells as 1. Results are representative of two independent experiments. AD vs PrEC, **P values ranged from

Supportive validation

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Experimental details: Figure 2 TPR proteins are significantly enhanced upon ectopic expression and change Hsp90 heterocomplex composition. A, SK-N-MC cells were transfected with plasmid expressing one of the TPR proteins, lysed after 48 h and levels of the respective TPR protein was determined by Western blot analysis. B, HEK-293 cells were transfected with FLAG tagged Hsp90 along with FKBP52 expressing plasmid or control plasmid. Hsp90 was precipitated from lysates and the levels of co-precipitated cofactors were determined by Western blot.

Supportive validation

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Experimental details: Figure 1 Over-expression of Cyp40 and FKBP51 in PCa specimens. ( a ) Total RNA was isolated from 8 normal and 10 malignant human prostate tissues, followed by real-time PCR using Cyp40-, FKBP51- and AR-specific primers and normalization to ribosomal 18S. RNA levels were expressed as relative to normal prostate tissue #1. *P

Supportive validation

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Experimental details: Figure 5 Down-regulation of Cyp40 inhibits AR activity. LNCaP cells were transiently transfected with Cyp40 siRNA or non-target control siRNA. Ninety-six hours later, the WCEs were prepared, and expression of Cyp40 was measured by Western blotting ( a ). LNCaP cells were transfected with Cyp40 siRNA or control siRNA, and the PSA-Luc reporter construct along with a beta-gal plasmid. Forty-eight hours later, cells were treated with or without R1881, CsA, CsA and R1881 for 24 h. Lysates were prepared and analyzed AR reporter gene activity ( b ). Results are means +- SEM of two independent experiments. *P

Supportive validation

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Experimental details: Figure 8 Down-regulation of Cyp40 predominantly inhibits androgen-dependent growth in PCa cells. AD LNCaP and AI C4-2 cells were transiently transfected with Cyp40-siRNA or control-siRNA. Six to 8 h after transfection, medium was changed. Twenty-four hours after transfection, cells were plated (1500 cells / well) in 96-well (cell proliferation assay) and 6-well plates (Western blotting). Seventy-two hours after transfection, LNCaP ( a ) and C4-2 ( c ) cells were treated with R1881 (0.1 and 1.0 nM) or vehiclel (0) for 24 h. Whole cell extracts were prepared, and expression of Cyp40 and actin (control) was measured by Western blotting. LNCaP ( b ) and C4-2 ( d ) cells were treated with R1881 (0.1 and 1.0 nM) or vehicle (0) alone for 3 days. Ninety-six hours after transfection, cell proliferation was determined by means of an MTT assay. Results are means +- SEM of two independent experiments, each performed in quadruplicate. LNCAP: pair-wise comparison of control siRNA vs Cyp40 siRNA at each condition, ***P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Cyp 40 is unable to compete the inhibitory effect of FKBP51. SK-N-MC cells were transfected with the MMTV-Luc reporter plasmid, the Gaussia-KDEL control plasmid, one of the plasmids expressing the HA-tagged GR or MR as indicated, and plasmids expressing FKBP51 and Cyp40 at the indicated amounts. After transfection, the cells were cultivated for 24 h in the presence of 10 nM cortisol (A) or 0.03 nM Fludrocortisol (B). Bar graphs indicate the relative reporter activity representing Firefly measurements normalized to Gaussia activities and presented as relative stimulation + S.E.M. of three independent experiments performed in triplicate. Lower panel of A displays immunoblots of cell extracts, probed with HA antibody demonstrating GR expression and the same membrane probed with FLAG antibody to detect overexpressed FKBP51 and Cyp40, and actin as control. In addition, antibodies directed against FKBP51 or Cyp40 were used to visualize the combined levels of endogenous and ectopic TPR protein.