PMC

(a) HEK293 cells were transiently transfected with HA–FNIP1 or empty vector (EV), (b) or HA–FNIP2 or empty vector (EV), and treated with indicated amounts of GB. (c) Induction of apoptotic markers in lysates from a and b were shown by immunoblotting using anti-cleaved caspase-3 and cleaved PARP antibodies. (d) FNIP1, FNIP2 or both were silenced by siRNA in HEK293 cells and then treated with indicated amounts of GB. Apoptotic markers were examined by immunoblotting using anti-cleaved caspase-3 and cleaved PARP antibodies.

(a) FLAG-tagged Hsp90α-N-, M-, and C-domains were transiently expressed and immunoprecipitated from HEK293 cells. Co-IP of endogenous FLCN and FNIPs were assessed by immunoblotting. Empty vector (EV) was used as a control. (b) HA-tagged FNIP1 domains were transiently expressed and isolated from HEK293 cells. Co-IP of endogenous FLCN and Hsp90 were assessed by immunoblotting. Empty vector (EV) was used as a control. (c) FLAG-tagged Hsp90α domains were transiently co-expressed with HA-tagged FNIP1-D in HEK293 cells. HA–FNIP1-D was immunoprecipitated and Hsp90α–FLAG co-IP was evaluated by immunoblotting. (d) Bacterially expressed and purified FNIP1-D and Hsp90α binding affinity measured by fluorescence anisotropy. The error bars represent mean ±s.d. of three independent experiments.

(a) Structure of human Hsp90α N-domain bound to ATP (PDB: 3T0Z). (b) HA–FNIP1, HA–FNIP2 or empty vector (EV) were transiently overexpressed in HEK293 cells. Lysates were incubated with ATP agarose. Hsp90 binding to ATP agarose was examined by immunoblotting. (c) Structure of human Hsp90α N-domain bound to GB (PDB: 3TUH). (d) cMyc–FNIP1, cMyc–FNIP2 or empty vector (EV) were transiently overexpressed in HEK293 cells. Lysates were incubated with indicated amounts of biotinylated GB followed by streptavidin agarose beads. Hsp90 was detected by immunoblotting. (e) Structure of human Hsp90α bound to related SNX2112 compound, tetrahydro-4H-carbazol-4-one (PDB: 3D0B). (f) Lysates from d were incubated with indicated amounts of biotinylated SNX2112 followed by streptavidin agarose beads. Hsp90 was detected by immunoblotting. (g) FNIP1 and FNIP2 were silenced by siRNA in HEK293 cells. Lysates were incubated with ATP agarose. Hsp90 binding to ATP agarose was examined by immunoblotting. NT represents non-targeting siRNA control pools. (h) Lysates from g were incubated with indicated amounts of biotinylated GB followed by streptavidin agarose beads. Hsp90 was detected by immunoblotting.

(a) Hsp90 was immunoprecipitated from HEK293 cells and LNCaP (prostate), T24 (bladder), MCF7 (breast), H1299 (lung) and HT29 (colorectal) cancer cell lysates using anti-Hsp90 or IgG (control), and immunoblotted with FNIP1 and FNIP2 antibodies to confirm protein interaction. Short and long exposures of the blots are shown. (b) Both FNIP1 and FNIP2 were silenced by siRNA in LNCaP, (c) T24, (d) MCF7, (e) H1299 and (f) HT29, and then treated with indicated amounts of GB. Apoptotic markers were shown by immunoblotting using anti-cleaved caspase-3 and cleaved PARP antibodies. Anti-cleaved caspase-7 antibody was only used for MCF7 cells.

(a) Hsp90α–FLAG was transiently expressed in HEK293 cells for 24 h followed by siRNA knockdown of FNIP1 and/or FNIP2. Hsp90α–FLAG was immunoprecipitated (IP) and co-IP of the co-chaperones was assessed by immunoblotting. Densitometry of the western blotting for FNIPs is represented as mean±s.d. A Student's t-test was performed to assess statistical significance (*P<0.05, **P<0.005 and ***P<0.0005). (b) HEK293 cells were transiently co-transfected with Hsp90α–FLAG and HA–FNIP1 or HA–FNIP2. Hsp90α–FLAG was isolated and co-IP of co-chaperones examined by immunoblotting. (c) HA–FNIP1, HA–FNIP1-D and HA–FNIP2 inhibited Hsp90α–HA ATPase activity after 30 min. Addition of 1.3 μM Aha1–FLAG stimulated the ATPase activity. All the data represent mean±s.d. A Student's t-test was performed to assess statistical significance (**P<0.005 and ****P<0.0001). (d) FNIP1 and Aha1 compete for binding to Hsp90α. FNIP1-D–His₆ was attached to Ni-NTA agarose and then incubated with Hsp90α. Ni-NTA agarose was then washed and incubated with the indicated amounts of Aha1–FLAG. (e) Aha1–FLAG attached to anti-FLAG M2 affinity gel was incubated with Hsp90α initially and then washed and incubated with indicated amounts of the FNIP1-D–His₆.

(a) BY4741 yeast strain expressing GAL1-LST4-GST was grown on either YPED (glucose) or YPGal (Galactose) media at 30 °C for 8 h. Endogenous yHsp90 immunoprecipitation (IP) and co-IP Lst4-GST was detected by immunoblotting. (b) In vitro ATPase activity of the yHsp90–His₆ with indicated amounts Lst4-GST. All the data represent mean±s.d. (c) PP30 strain expressing either yHsp90 or Hsp90α as the sole copies of Hsp90 were transformed by GAL1–FLAG–FNIP1 or GAL1–FLAG–FNIP2. Cells were spotted at 1:10 dilution series on YPED or YPGal media. Plates were incubated at 30 °C for 2 days. (d) Yeast strains in i were grown on YPGal liquid media for 12 h and the expression of FLAG–FNIP1 and FLAG–FNIP2 were examined by immunoblotting.

(a) Clear cell renal cell carcinoma (ccRCC), (b) Papillary type I, (c) Papillary type II, (d) Oncocytoma (Tumours, T) and adjacent normal tissues (Normal, N) were stained with haematoxylin and eosin (H&E). Proteins were also extracted from these tumours and adjacent normal tissues and incubated with indicated amounts of biotinylated GB followed by streptavidin agarose beads. Hsp90 was detected by immunoblotting. Expression of FNIP1 and FNIP2 in these samples was also detected by immunoblotting. (e) Hsp90 immunoprecipitated from tumours (T) and adjacent normal tissues (N) in a–d. Co-IP of FNIPs and Aha1 was examined by immunoblotting. (f) Lysates from normal tissues in a–d were incubated with or without 100 ng of pure FNIP1-D–His₆ for 10 min. Hsp90 was immunoprecipitated and co-IP of FNIP1-D–His₆ and Aha1 were shown by immunoblotting.

(a) FNIP1 and FNIP2 were isolated from HEK293 cell lysates using anti-FNIP1, anti-FNIP2 or IgG (control) and immunoblotted with indicated antibodies to confirm protein interaction. (b) HA–FNIP1 and HA–FNIP2 were transiently co-expressed with either cMyc–FNIP1 or cMyc–FNIP2 in HEK293 cells. cMyc–FNIP1 and cMyc–FNIP2 were first isolated followed by IP of HA–FNIP1 and HA–FNIP2 from the same samples. The quality of HA–FNIP1:cMyc–FNIP1 and HA–FNIP2:cMyc–FNIP2 homodimers and HA–FNIP1:cMyc–FNIP2 heterodimer were assessed by immunoblotting. Co-IP of Hsp90 and FLCN was also assessed by western blotting. (c) HEK293 cells were treated with 1 μM GB for the indicated times, and FNIP1 and FNIP2 protein levels were determined by immunoblotting. Akt and Phospho-S473-Akt were used as positive controls. (d) FLAG–FNIP1 interacts with Hsp90α–His₆ in vitro. Bacterially expressed and purified Hsp90α–His₆ was bound to Ni-NTA agarose and then incubated with 10 ng pure FLAG–FNIP1. Hsp90α–His₆ pulldown and FLAG–FNIP1 co-pulldown were assessed by immunoblotting. (e) FLAG–FNIP1 interacts with FLCN–His₆ in vitro. Bacterially expressed and purified FLCN–His₆ was bound to Ni-NTA agarose and then incubated with 10 ng pure FLAG–FNIP1. FLCN–His₆ pulldown and FLAG–FNIP1 co-pulldown were assessed by immunoblotting. (f) FLCN–His₆, FLAG–FNIP1 and Hsp90α tri-complex in vitro. Bacterially expressed and purified FLCN–His₆ was bound to Ni-NTA agarose and followed by incubation with 10 ng FLAG–FNIP1 and then 10 ng untagged Hsp90α in vitro. FLCN–His₆ pulldown, and FLAG–FNIP1 and Hsp90α co-pulldown were examined by immunoblotting.

(a) Effect of siRNA knockdown of FNIP1 and FNIP2 on Hsp90 clients. Stability and activity of the indicated clients were assessed by immunoblotting. Densitometry of the western blotting for FNIP1 and FNIP2 is represented as mean±s.d. A Student's t-test was performed to assess statistical significance (**P<0.005 and ***P<0.0005). (b) Transient overexpression of cMyc-tagged FNIP1 or FNIP2 in HEK293 cells and their impact on levels of Hsp90 clients was assessed by immunoblotting. Empty vector (EV) was used as a control. (c) HEK293 cells were co-transfected with CFTR and indicated HA–FNIP1 and HA–FNIP2 constructs. After 24 h, CFTR and FNIPs–HA were detected by immunoblotting; GAPDH was used as loading control. Empty vector (EV) was used as a control. (d) In vitro ATPase activity of Hsp90α–FLAG isolated from PC3 cells. Inhibitory effects of purified HA–FNIP1, FNIP1-D–HA or HA–FNIP2 on ATPase activity of Hsp90α–FLAG. All the data represent mean±s.d. A Student's t-test was performed to assess statistical significance (***P<0.0005 and ****P<0.0001). (e) ATPase activity of Hsp90α–FLAG from d was inhibited by addition of 10 μM GB. All the data represent mean±s.d. A Student's t-test was performed to assess statistical significance (****P<0.0001).

(a) FLAG–FLCN was expressed and isolated from HEK293 cells. Profile of interacting proteins determined by MALDI–time of flight. Red nodes represent chaperones and co-chaperones, blue nodes are chaperonins and green nodes are splicing factors and ribosomal proteins. (b) FLCN was isolated from HEK293 cell lysates using anti-FLCN or IgG (control) and immunoblotted with indicated antibodies to confirm protein interactions. (c) HEK293 cells were transiently transfected with FLAG–FLCN or empty vector control (EV), immunoprecipitated and immunoblotted with indicated antibodies to confirm interacting proteins. (d) HEK293 cells were treated with 10 μM of the Hsp70 inhibitor JG-98 at the indicated time points. FLCN protein stability in soluble and insoluble fraction was assessed by immunoblotting. (e) HEK293 cells were treated with 1 μM GB at the indicated time points. FLCN protein stability was assessed by immunoblotting. Akt and Phospho-S473-Akt were used as positive controls. (f) Hsp90α–FLAG was transiently expressed in HEK293 cells. Cells were treated with 1 μM GB for the indicated times. Hsp90α–FLAG was immunoprecipitated and co-IP of FLCN was examined by immunoblotting. (g) HEK293 cells were treated with 50 nM of the proteasome inhibitor bortezomib (BZ) for the indicated times. FLCN protein levels were evaluated at the indicated time points by immunoblotting (upper blots). HEK293 cells were also treated with 1 μM GB for 1 h before addition of 50 nM BZ. Immunoblotting was used to evaluate the FLCN level for the indicated time points (lower blots). (h) Empty vector (EV) or FLAG–FLCN was used to transiently transfect HEK293 cells for 24 h then treated for 4 h with either 50 nm BZ or 1 μM GB. FLAG–FLCN was immunoprecipitated and ubiquitination was examined by immunoblotting with a pan-anti-ubiquitin antibody.