Fig 1: Hsp90 can stabilize WTAP on the level of protein and mRNA. a OCI-Ly10, OCI-Ly19, SU-DHL2 and SU-DHL4 were cultured with different PU-H71 concentrations for 24 h. WTAP protein level was detected by western blot. b Cells treated with PU-H71 (1 μM) for various times, protein expression level were assessed by western blotting. c mRNA levels of WTAP were checked by qRT-PCR. Results are expressed as fold difference compared to baseline (time 0 h) and were normalized to GAPDH. Experiments were performed three times, each with duplicate qPCR measurements. Error bars represent the s.e.m. from three replicates. d Cells were pretreated with 100 nM Bortezomib, an inhibitor for 26 s proteasome, or DMSO only for 2 h. Then cells were cultured in the presence of 1 μM PU-H71 or DMSO for 6 h. WTAP expression was checked by western blotting. e HEK293T cells were transfected with pcDNA3.1-WTAP and HA-Ub, then treated with PU-H71 or DMSO for 24 h. Cell were collected to add anti-WTAP, and ubiquitination level were check. f, g Cells were pretreated with 1.5 μM PU-H71 or DMSO for 24 h, then cultured with Etoposide (10 μM) for another 24 h. The apoptosis rates were detected by flow cytometry. These experiments were repeated three times
Fig 2: Interaction between Hsp90 and Erk1/2 during human sperm capacitation. a. Hsp90 was detected in sperm lysates using western blotting and Co-IP with non-immune mouse IgG or antibodies against Erk1/2. No interaction was observed between non-immune mouse IgG and Hsp90, whereas a clear interaction was evident between Hsp90 and Erk1/2. 17-AAG significantly disrupted the interaction between Hsp90 and Erk1/2. b. The relative association of Hsp90 and Erk1/2 with or without 5 µM 17-AAG during human sperm capacitation
Fig 3: WTAP forms a complex with BCL6 via Hsp90. a OCI-Ly10 and OCI-Ly19 nuclear and cytoplasmic extracts were detected by Western blotting. b Co-localization of endogenous WTAP (red) and BCL6 (green) in DLBCL cells was detected by antibody stainings and confocal microscopy. When indicated, cells had been treated with PU-H71 (1 µM). Co-localization is shown by merge (yellow or orange). Nuclei were counterstained with DAPI (blue). c OCI-Ly19 cell lysates were supplemented with anti-WTAP, anti-BCL6 and negative control IgG antibodies. The protein complex was assessed against indicated antibodies. d Exogenous expression of WTAP and BCL6 was achieved by expression plasmids (pcDNA3.1-WTAP and BCL6-His) transfected into HEK293T cells and cultured for 24 h. PU-H71 (1.5 µM) or DMSO was added for another 24 h. Whole cell lysates were precipitated by anti-WTAP antibodies. WTAP, BCL6 and Hsp90 protein level were detected by the respective antibodies. LaminA/C as a nuclear protein served as a negative control. e, f GST pull-down assay to determine if WTAP interacts with BCL6 via Hsp90, either with GST-WTAP and BCL6-His only (e) or in the presence of whole cell extracts (f)
Fig 4: Interaction between Hsp90 and its co-chaperone Cdc37 in human sperm. a. Hsp90 was detected in sperm lysates using western blotting and Co-IP with non-immune mouse IgG or antibodies against Cdc37. No interaction was observed between non-immune mouse IgG and Hsp90, whereas a clear interaction was evident between Hsp90 and Cdc37. 17-AAG disturbed the formation of the Hsp90-Cdc37 complex. Results are representative of three independent experiments using samples from three different individuals. b. The relative association of Hsp90 and Cdc37 with or without 5 µM 17-AAG during human sperm capacitation
Fig 5: Producing functional myosin in insect cells. a Overview of constructs used for expression studies in insect cells. Western blot analysis of co-expression of NMY-2 (b) or MHC-B (c) with indicated chaperones in Hi5 insect cells. Whole cell lysates (WCL) and soluble fractions (SOL) are shown. 1/3rd of the WCL was applied with respect to the SOL fraction. (SBD–substrate binding domain, NBD–nucleotide binding domain, NTD–N-terminal domain, MD–middle domain, CTD–C-terminal domain). d Pull-down (PD) of Strep-tagged UNC-45 constructs from insect cells. Input samples and elutions were probed with Hsp70, Hsp90, and Strep-tag antibodies. e Upper panel: SEC traces of MHC-B purified from co-expression with the indicated C. elegans chaperones. Lower panel: SDS-PAGE gels showing the elution after NiNTA and indicated SEC fractions
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