PA3-013

antibody from Invitrogen Antibodies

Targeting: HSP90AA1 FLJ31884, Hsp89, Hsp90, HSP90N, HSPC1, HSPCA

Provider product page for PA3-013

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [53]
Comments [0]
Validations
Immunocytochemistry [2]
Other assay [32]

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Product number: PA3-013 - Provider product page
Provider: Invitrogen Antibodies
Product name: HSP90 alpha Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA3-013 detects heat shock protein 90 (HSP90) from human, monkey, mouse, sheep (adrenal) and rat tissues and cells. This antibody does not detect HSP84. PA3-013 has been successfully used in Western blot, immunofluorescence, immunohistochemistry (paraffin) and immunoprecipitation procedures. By Western blot, this antibody detects an ~86 kDa protein representing HSP86 from HeLa cell lysate. Immunofluorescence staining of HSP86 in HeLa cells with PA3-013 yields a pattern consistent with cytoplasmic staining. Immunoprecipitation experiments with this antibody suggest that HSP86 exists primarily as homodimers in HeLa cells. Furthermore, the antibody is capable of precipitating HSP86 that is complexed with other proteins such as the aryl hydrocarbon (Ah) receptor. The PA3-013 immunogen is a synthetic peptide corresponding to residues P(2) E E T Q T Q D Q P M(12) of mouse HSP86. The N-terminal regions of HSP84 and HSP86 show the largest difference in amino acid sequence.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

HSP90AA1 protein structure - PA3-013 shown in red.

Supportive validation

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Experimental details: Immunofluorescence analysis of HSP90 alpha was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with HSP90 alpha Polyclonal Antibody (Product # PA3-013) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasm and weak Nucleus localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Heat Shock Protein 86 (HSP86, green) in HeLa cells and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a HSP86 polyclonal antibody (Product # PA3-013), at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat-anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

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Experimental details: Figure 5 ARD1-mediated AR acetylation drives ligand-induced AR-HSP90 dissociation ( A ) His-AR and Flag-ARD1 were co-transfected in 293T cells. In vitro interaction of AR, ARD1, and HSP90 were analyzed by sequential immunoprecipitation and immunoblotting with antibodies as indicated. ( B ) In vivo interaction of endogenous ARD1, AR, and HSP90 in LNCaP cells by immunoprecipitation and immunoblotting using antibodies as indicated. ( C ) Immunoblot of interaction between AR-WT and Myc-ARD1-WT transfected in Cos-7 cells in the presence or absence of R1881, immunoprecipitated with anti-Myc antibody. ( D ) AR and Flag-HSP90 were co-transfected with increased amounts of Myc-ARD1 in Cos-7 cells. Ligand-induced and ARD1 dose dependent AR acetylation were measured by immunoprecipitation with an anti-AR antibody and immunoblotted with an antibody specifically against acetylated lysine, while AR-HSP90 dissociation was measured by immunoprecipitation with an anti-Flag antibody and HSP90 co-immunoprecipitated AR was observed by Western blot. ( E ) AR and Flag-HSP90 co-transfected in Cos-7 cells in the presence or absence of R1881 were immunoprecipitated (first IP) with an anti-Flag antibody and immunoblotted using an antibody against AR (left two lanes). A secondary immunoprecipitation was performed on the supernatant after first IP using antibody specifically against acetylated lysine and immunoblotted with an anti-AR antibody (right two lanes). ( F ) Flag-HSP90 co-transfected with AR-WT,

Supportive validation

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Experimental details: Figure 1 Functional disruption of the Hsp90alpha gene in the mouse. (A) Schematic representation of the disruption of the mouse Hsp90aa1 gene by insertion of a gene trap (GT) in intron 10 (in mutant mouse line 1). Open and black boxes indicate non-coding and coding exons, respectively. The gene trap consists of a splice acceptor (SA), a beta-galactosidase-neomycin resistance fusion (betaGeo) and an SV40 polyadenylation site. (B) Immunoblots showing the expression of Hsp90alpha and Hsp90beta in a panel of tissues in wild-type (WT) and mutant (gt/gt) mice (of 136 days postnatal). The Ponceau S staining of the immunoblot filter gives an indication of protein loading.

Supportive validation

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Experimental details: Figure 6 Immunoblot analysis of a panel of Hsp90- and Hsp70-related proteins. The results shown are from a representative analysis of testis extracts from wild-type and mutant animals from P12 until completion of the first wave of spermatogenesis at P44. GAPDH serves as loading control. Note that the order of the sample pairs is reversed for the P16 and P18 samples at the center of the blots (area highlighted by hairlines).

Supportive validation

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Experimental details: Figure 6 Experimental validation of the involvement of Aha1 in nucleocytoplasmic transport. (A) PPI map of the proteins (red nodes) in the GO module ""nucleocytoplasmic transport"" of the Aha1-Hsp90 PPI subset of Figure 5 . It contains potential and known (dashed and full line edges, respectively) Aha1 interactions. The Hsp90 client protein GR was also integrated into the predicted PPI (upper panel). The co-immunoprecipitation assays of panel B allowed the experimental confirmation and new demonstration of PPIs as indicated by red and green edges, respectively (lower panel). (B) Co-immunoprecipitation experiment demonstrating interactions between Aha1 or exportin-1 (XPO1) and components of the GO module ""nucleocytoplasmic transport"" shown in panel A. Flag-tagged Aha1, exportin-1, and GPR30 as an unrelated control protein were exogenously expressed in 293T cells. IPO4, importin-4; KPNA5, importin-alpha6. (C) and (D) Nuclear localization of GR in mouse fibroblasts with and without Aha1. Panel C shows representative micrographs of the localization of Tom.GR with our without treatment with dexamethasone (Dex) for 40 min, and an immunoblot on the right verifying the absence of Aha1 in the Aha1- fibroblasts. Panel D shows the nuclear accumulation of Tom.GR over time in wild-type (^) and Aha1- (V) cells. Nuclear localization of GR was initiated at time zero with the addition of 10 nM dexamethasone. Data points are the means with standard errors of three independent experim

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Heat Shock Protein (HSP86) was performed on HeLa cells. Antigen-antibody complexes formed by incubating 500 µg whole cell lysate with 2 µg of HSP86 polyclonal antibody (Product # PA3-013) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µL Protein A/G Plus Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a HSP86 polyclonal antibody (Product # PA3-013) at a dilution of 1:1000 overnight rotating at 4C. The membrane was washed in TBST, and probed with Clean-Blot IP detection Reagent (Product # 21230) at a dilution of 1:1000 for at least 1 hour. Chemiluminescent detection was performed using Pierce SuperSignal West Dura (Product # 34075).

Supportive validation

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Experimental details: Fig. 1 HSP90A is required for multi-aggressive properties of immune-edited tumor cells. a HSP90A protein level in CaSki P0 and P3 cells was determined by Western blot. beta-ACTIN was included as an internal loading control. Numbers below blot images indicate the expression as measured by fold change. b HSP90AA1 mRNA level in CaSki P0 and P3 cells was determined by qRT-PCR. c-e CaSki P0 and P3 cells were transfected with siGFP or siHSP90AA1-#1. c The frequency of apoptotic (active caspase-3 + ) cells in the MART-1 peptide pulsed cells after incubation with or without MART-1-specific CTLs at a 1:1 ratio for 4 h was estimated by flow cytometry analysis. d and e Cells were treated with indicated concentrations of cisplatin d and irradiation e . The percentage of viable cells was determined by trypan blue exclusion assay at 24 h after either cisplatin treatment or irradiation. * p < 0.01, ** p < 0.001, and *** p < 0.0001. f CaSki P0 and P3 cells were transfected with siGFP, siHSP90AA1-#1, or siHSP90AA1-#2. Sphere-forming capacity of the cells in low-density suspension culture. All experiments were performed in triplicate. The p -values by two-tailed Student's t test b or two-way ANOVA c-f are indicated. NS, not significant. Data represent the mean +- SD. Source data are provided as a Source Data file.

Supportive validation

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Experimental details: Fig. 2 HSP90AA1 expression is directly regulated by NANOG. a and b CaSki P3 cells were transfected with siRNA-targeting GFP or NANOG. a Levels of NANOG and HSP90A protein were analyzed by Western blot. b HSP90AA1 mRNA expression was analyzed by qRT-PCR. c and d CaSki P0 cells were stably transfected with empty vector (no insert) or NANOG. c Levels of NANOG and HSP90A protein were analyzed by Western blot. d HSP90AA1 mRNA expression was analyzed by qRT-PCR. e and f HEK293 cells were transfected with empty vector (no insert), FLAG-NANOG wild type (NANOG WT) or FLAG-NANOG mutant (NANOG MUT). e Levels of HSP90A and FLAG-NANOG proteins were proved by Western blot. f HSP90AA1 mRNA expression was analyzed by qRT-PCR. g Diagram of HSP90AA1 promoter region (-1322 to +190) containing NANOG binding element. The arrows indicate ChIP amplicon corresponding to -1048 to -899. h Luciferase assay in HEK293 cells transfected with the pGL3-HSP90AA1 WT or MUT plasmid, together no insert, NANOG WT or NANOG MUT plasmids. i Chromatin immunoprecipitation assay was carried out using HEK293 cells transfected with FLAG-NANOG. Cross-linked chromatin was immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated DNAs were amplified with PCR primers specific for the HSP90AA1 promoter region indicated above. Mouse IgG was used as a negative control. The input represents 2% of the total chromatin. j ChIP assay was carried out using CaSki P0 and P3 cells. Cross-linked chromatin was immunoprecipitated w

Supportive validation

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Experimental details: Fig. 3 NANOG-HSP90A axis is conserved across various human cancer types. a Protein levels of NANOG and HSP90A in various human cancer cells were determined by immunoblotting. This experiment was performed in triplicate. b Correlation between NANOG and HSP90A level normalized by beta-ACTIN level in various human cancer cells (Spearnan r = 0.7741, p = 0.0028). c Representative images of IHC staining of HSP90A in cervical tissue from normal ( n = 328), LGCIN ( n = 65), HGCIN ( n = 160), and cervical carcinoma ( n = 151) patients. Scale bar shown is 250 um. LGCIN low-grade CIN; HGCIN high-grade CIN. d Correlation between NANOG and HSP90A in patients with cervical cancer (Spearnan r = 0.746, p < 0.001). e and f Combined level of NANOG + /HSP90A + was significantly associated with large-sized tumors e and chemoradiation resistance f in patients with cervical cancer. Chemoradiation resistance was calculated only cases with available information of chemoradiation response. g Patients with NANOG + /HSP90A + level displayed worse disease-free survival ( p = 0.007) than patients with NANOG - /HSP90A - level. Cut-off value of NANOG + and HSP90A + are 160 and 127, respectively. The p -values were determined by Mann-Whitney U test e and f and Log-rank (Mantel-Cox) test g or spearman correlation ( r ) b and d . In the box plots, the top and bottom edges of boxes indicate the first and third quartiles, respectively; the center lines indicate the medians; and the ends of whiskers indicate the

Supportive validation

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Experimental details: Fig. 4 HSP90A contributes to NANOG-induced AKT activation through TCL1A-stabilization. a-e CaSki-NANOG cells were transfected with siGFP or siHSP90AA1-#1. a Levels of HSP90A, TCL1A, pAKT, AKT, MCL1, and Cyclin A were analyzed by immunoblotting. Graph depicts the experimental quantitation based on at least three independent experiments. ** p < 0.001, *** p < 0.0001 by two-tailed Student's t test. NS not significant. b The cells were treated with cycloheximide (CHX) for the indicated times. Cell lysates were subjected to immunoblotting with anti-TCL1A antibodies. Graph represents the means +- SD of three quantified data, after normalization to the corresponding beta-ACTIN level. c The cells were treated with or without MG132 (10 muM) for 8 h. Cell lysates were subjected to immunoblotting with anti-HSP90A and anti-TCL1A antibodies. d The cells, transfecte d with indicated plasmids, were treated with MG132. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with the anti-HA antibody. The input represents 5% of total proteins. e siHSP90AA1-transfect e d CaSki-NANOG cells were transfected with TCL1A-Myc constructs. Levels of HSP90A, TCL1A, pAKT, and AKT were analyzed by immunoblotting. f HEK293 cells were transfected with TCL1A-Myc. g TCL1A-Myc-transfected HEK293 cells were treated with DMSO or AUY-922 as indicated. f and g Cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blotting using anti-Myc and anti-HSP90A antibodies.

Supportive validation

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Experimental details: Fig. 5 HSP90A inhibition reduces multi-aggressive properties of NANOG high tumor cells. a-e CaSki P3, MDA-MB231 P3, SiHa, and HCT116 cells were treated with DMSO or AUY-922. a Levels of HSP90A, TCL1A, pAKT, AKT, MCL1, Cyclin A, and beta-ACTIN were proved by Western blot. beta-ACTIN was included as an internal loading control. Numbers below blot images indicate the expression as measured by fold change. Graph depicts the experimental quantitation based on at least three independent experiments. b Flow cytometry analysis of the frequency of apoptotic (active caspase-3 + ) cells in the cells after intracellular delivery of granzyme B for 4 h. c and d Cells were treated with indicated concentrations of cisplatin c and irradiation d . The percentage of viable cells was determined by trypan blue exclusion assay at 24 h after either drug challenge or irradiation. e Sphere-forming capacity of the cells in low-density suspension culture. All experiments were performed in triplicate. The p -value by two-tailed Student's t test a , one-way ANOVA b and e or two-way ANOVA c and d are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data represent the mean +- SD. Source data are provided as a Source Data file.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Proteolysis of Hsp90from TnT reticulocyte lysate incubated underconditions of protein synthesis with (A) vehicle (1% DMSO) and 5 mMnovobiocin and (B) vehicle (1% DMSO) or 50 nM or 50 muM cruentarenA. Antibodies specific to either the N- or C-terminus of Hsp90 wereused to identify the Hsp90 fragments produced in the presence of increasingamount of trypsin.