PA3-013

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Heat Shock Protein 86 (Hsp86) was performed by loading 2-fold serial dilutions of HeLa cell lysate, starting at 10 ug, per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Hsp86 polyclonal antibody (Product # PA3-013) at a dilution of 1:1000 overnight at 4ºC on a rocking platform, washed in TBS-0.1%Tween-20, and probed with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31466) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using ECL Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Hsp86 and Hsp84, using Product # PA3-013 and Product # PA3-012, respectively

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of epidermoid carcinoma (A431) cells using Heat Shock Protein 86 Polyclonal Antibody (Product # PA3-013). Decreasing amounts of A431 whole cell lysates were probed with PA3-013 at a dilution of 1:2000 followed by an anti-rabbit IgG secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (Product # 34087). Lanes 1 and 2 are control samples of purified, recombinant Hsp86 (rHsp86) and Hsp84 (rHsp84), respectively. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Heat Shock Protein 86 (Hsp86) was performed by loading 2-fold serial dilutions of HeLa cell lysate, starting at 10 ug, per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Hsp86 polyclonal antibody (Product # PA3-013) at a dilution of 1:1000 for 1 hour at room temperature on a rocking platform, washed in TBS-0.1%Tween-20, and probed with an AP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31340) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using Lumi-Phos Substrate (Product # 34150).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HSP90 alpha Polyclonal Antibody(Product # PA3-013) and a 80kDa band corresponding to HSP90 alpha was observed across cell lines and tissue extracts tested. Whole Cell Extract-WCL (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), LNCaP (Lane 3), NIH/3T3 (Lane 4) and tissue extracts of Mouse Testis (Lane 5) and Rat Testis (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Heat Shock Protein 86 (HSP86) was performed by loading 50 µg of the indicated whole cell lysates and 15 µL of PageRuler Prestained Protein Ladder (Product # 26616) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were 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 at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a goat anti-rabbit IgG HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissues. 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 Heat Shock Protein 90 (86) (Product # PA3-013) 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 normal deparaffinized Human kidney tissue tissues. 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 Heat Shock Protein 90 (86) (Product # PA3-013) 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 normal deparaffinized Human tonsil tissue tissues. 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 Heat Shock Protein 90 (86) (Product # PA3-013) 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 differentiated colon adenocarcinoma from paraffin-embedded human colon tissue sections. To expose target proteins, heat-induced epitope retrieval was performed using 10mM sodium citrate (pH 6.0) buffer for 20 minutes at 95ºC. Following antigen retrieval, tissues were blocked in 3% BSA (Product # 37525) in PBST for 30 minutes at room temperature and then probed with a Heat Shock Protein 86 (Hsp86) polyclonal antibody (Product # PA3-013) at a dilution of 1:100 for 1 hour in a humidified chamber (right panel). As a negative control, the primary antibody was eliminated from the staining procedure (left panel). Tissues were washed extensively with PBS/0.025% Tween-20 (Product # 28314) and endogenous peroxidase activity quenched with Peroxidase Suppressor (Product # 35000) for 30 minutes at room temperature. Detection was performed using an HRP-conjugated goat anti-rabbit IgG-HRP secondary antibody (Product # 31466) at a dilution of 1:250 followed by colorimetric detection using Metal Enhanced DAB Substrate Kit (Product # 34065). Tissues were counterstained with hematoxylin and prepped for mouting. Images were taken on a Zeiss Axiovision microscope at 40X magnification (x1.6 Optovar).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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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.