Journal: Molecular Biology of the Cell

Article Title: Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells

doi: 10.1091/mbc.E12-12-0892

Figure Lengend Snippet: Knockdown of Hsp90 cochaperones reduces efficiency of RNAi. (A) HeLa cells were harvested 72 h after transient cotransfection with pLKO.1 vectors expressing shRNAs against hAgo2, FKBP4, Cdc37, Aha1, p23, or a nonsilencing control (nsc) as a control and a GFP-based reporter for RNAi activity (GFP-let7a) or the control (GFP-let7XX). Lysates were subjected to SDS–PAGE and immunoblotted for GFP and actin as a loading control. (B) GFP expression was quantitated using Odyssey software and normalized to actin. RNAi efficiency was calculated by determining the relative GFP expression between cells expressing GFP-let7a or GFP-let7XX. The efficiency of RNAi-mediated silencing was calculated independently for each shRNA, and the pLKO.1-nsc–transfected sample was set to 100%. Error bars represent SE, and n values are indicated below the axis. * p < 0.05; ** p < 0.01.

Article Snippet: Other primary antibodies were from the following sources: rabbit polyclonal anti-GFP from L. Berthiaume (University of Alberta, Edmonton, Canada); mouse monoclonals anti-Dicer (ab14601), anti-hAgo2 (ab57113), anti-FKBP4 (ab59460), and anti-p23 (ab2814), and rabbit polyclonal anti-FKBP4 (ab97306) from Abcam (Cambridge, MA); goat polyclonal anti-HSP90 (sc-1055) from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal anti-Hsp70 (SPA-810) from Enzo Life Sciences (Farmingdale, NY); mouse monoclonals anti-Cdc37 (MA3-029) and anti-p23 (MA3-414) from Thermo Fisher Scientific (Waltham, MA).

Techniques: Cotransfection, Expressing, Activity Assay, SDS Page, Software, shRNA, Transfection

Journal: Molecular Biology of the Cell

Article Title: Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells

doi: 10.1091/mbc.E12-12-0892

Figure Lengend Snippet: Knockdown of Hsp90 cochaperones does not affect the level of RNAi effector proteins. (A) HeLa cells were harvested 72 h after transient cotransfection with pLKO.1 vectors expressing shRNAs against hAgo2, FKBP4, Cdc37, Aha1, p23, or a nonsilencing control (nsc) as a control. Lysates were subjected to SDS–PAGE, and the levels of the RNAi effector proteins Dicer and hAgo2 were determined by immunoblot analysis. (B) Lysates from A were immunoblotted for the targeted cochaperones FKBP4, Cdc37, Aha1, and p23. (C) Signal intensities for FKBP4, Cdc37, Aha1, and p23 were quantitated for each knockdown. The bar graph indicates the average level of expression of the shRNA-targeted protein remaining 72 h posttransfection relative to the nonsilencing control.

Article Snippet: Other primary antibodies were from the following sources: rabbit polyclonal anti-GFP from L. Berthiaume (University of Alberta, Edmonton, Canada); mouse monoclonals anti-Dicer (ab14601), anti-hAgo2 (ab57113), anti-FKBP4 (ab59460), and anti-p23 (ab2814), and rabbit polyclonal anti-FKBP4 (ab97306) from Abcam (Cambridge, MA); goat polyclonal anti-HSP90 (sc-1055) from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal anti-Hsp70 (SPA-810) from Enzo Life Sciences (Farmingdale, NY); mouse monoclonals anti-Cdc37 (MA3-029) and anti-p23 (MA3-414) from Thermo Fisher Scientific (Waltham, MA).

Techniques: Cotransfection, Expressing, SDS Page, Western Blot, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Chronic treatment with the complex I inhibitor MPP + depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)

doi: 10.1074/jbc.RA119.010474

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Densitometry data were normalized to loading control, and the half-lives of PINK1 isoforms were estimated by fitting a one-phase exponential decay curve using Prism 8 software (GraphPad, San Diego, CA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Catalog number Source Dilution PINK1 Rabbit BC100-494 Novus Biological 1:1000 TOM20 Rabbit Sc11415 Santa Cruz 1:10,000 β-ACTIN Mouse A5316 Sigma 1:2000 HSP60 Mouse 611563 BD Transduction 1:1000 BAG6 Mouse Sc365928 Santa Cruz 1:1000 BAG5 Rabbit NB100-56091 Novus Biological 1:1000 BAG2 Mouse Sc101216 Santa Cruz 1:1000 HSP90 Rabbit 4877 Cell Signaling 1:1000 CDC37 Rabbit 3604 Cell Signaling 1:1000 CHIP Rabbit Sc66830 Santa Cruz 1:1000 GAPDH Rabbit Ab37168 Abcam 1:10,000 GFP Rabbit A6455 Invitrogen 1:10,000 V5 Mouse Sc271944 Santa Cruz 1:1000 FLAG Rabbit F7425 Sigma 1:1000 β-Tubulin Rabbit Ab6046 Abcam 1:5000 Open in a separate window List of antibodies used in the study

Techniques: Transduction

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

doi: 10.1016/j.ymthe.2022.02.009

Figure Lengend Snippet: Figure 1. F3-T3 shows a strong interaction with HSP90 and CDC37 (A) Schematic diagram of the methodology used for the identification of proteins that potentially interact with F3-T3. Cell lysates from U-251 MG cells stably expressing HA- F3-T3 and HA-FGFR3 underwent immunoprecipitation (IP) with anti-HA antibody and proteins were analyzed using 2D-LC/MS. (B) Schematic diagram of F3-T3, WT FGFR3, and truncated FGFR3 proteins. (C and D) Lysates from U-251 MG cells stably expressing EV, F3-T3, FGFR3, and tFGFR3 were immunoprecipitated with anti-HSP90 or anti- CDC37 antibodies followed by WB as indicated. The arrow indicates the specific CDC37 band. (E) U-251 MG cells stably expressing HA-EV and HA-F3-T3 were treated for

Article Snippet: Supernatants were collected after centrifuging at 14,000 x g for 15 min at 4 C. IP was performed by incubating cellular lysate supernatants with either isotype control antibody- or anti-HA antibody-conjugated magnetic beads (Cell Signaling Technology, 8726 and 11846), or with Santa Cruz mouse monoclonal anti-HSP90 (sc-13119) or CDC37 (sc-17758) antibodies followed by protein A agarose (Santa Cruz, sc-2001) or protein G plus agarose (Santa Cruz, sc-2002).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

doi: 10.1016/j.ymthe.2022.02.009

Figure Lengend Snippet: Figure 4. Targeting the HSP90-cdc37 system inhibits F3-T3 mediated signaling and oncogenic potency (A) U-251 MG cells stably expressing F3-T3 were exposed for 6 h to 200 nM of HSP90 inhibitors (onalespib and 17-AAG) or FGFR inhibitors (BGJ398 and PD173074) and lysates were immunoblotted with the indicated antibodies. (B) U-251 MG cells stably expressing F3-T3 were exposed for 6 h to 200 nM onalespib, 5 mg/mL brefeldin A (BFA) or 10 mg/mL of tunicamycin and lysates were immunoblotted with the indicated antibodies. (C) U-251 MG cells stably expressing F3-T3 were cultured in the absence or presence of FGF9 with or without 200 nM onalespib or 5 mg/mL BFA for 6 h and lysates were immunoblotted with the indicated antibodies. (D) U-251 MG cells stably expressing EV or F3-T3 were exposed to increasing concentrations of onalespib, 17-AAG, BGJ398, PD173074, or Vec control (DMSO) for 72 h, after which relative cell numbers were estimated using the CCK8 assay. A representative plot from 1 of 3 experiments is shown; means ± SEMs. (E) Mean IC50 of onalespib, 17-AAG, BGJ398, and PD173074 in U-251 MG cells stably expressing EV or F3-T3 from 3 independent experiments as illustrated in (D) and analyzed by 1-way ANOVA with Tukey’s post-test; *p < 0.05, ***p < 0.001 (selectively

Article Snippet: Supernatants were collected after centrifuging at 14,000 x g for 15 min at 4 C. IP was performed by incubating cellular lysate supernatants with either isotype control antibody- or anti-HA antibody-conjugated magnetic beads (Cell Signaling Technology, 8726 and 11846), or with Santa Cruz mouse monoclonal anti-HSP90 (sc-13119) or CDC37 (sc-17758) antibodies followed by protein A agarose (Santa Cruz, sc-2001) or protein G plus agarose (Santa Cruz, sc-2002).

Techniques: Stable Transfection, Expressing, Cell Culture, Control, CCK-8 Assay

Journal: ACS pharmacology & translational science

Article Title: Stabilization of Cyclin-Dependent Kinase 4 by Methionyl-tRNA Synthetase in p16 INK4a -Negative Cancer.

doi: 10.1021/acsptsci.8b00001

Figure Lengend Snippet: Figure 5. MRS facilitates CDK4−HSP90 complex formation and stabilizes CDK4. (a) H460 cells transfected with either si-Control or si-MRS were treated with a proteasome inhibitor (MG132). Relative levels of CDK4 were quantified using ImageJ. (b) MRS, CDC37, HSP90, and CDK4 were transfected into H460 cells, and their interaction was investigated. (c) Association of MRS with CDK4, HSP90, or CDC37 was investigated via in vitro pull-down assay. (d) Effect of geldanamycin (1 μM) on CDK4 in MRS-overexpressing H460 cells. (e) Effect of MRS on the interaction between CDK4 and HSP90 measured by reconstitution of nanoluciferase luminescence. (f) H460 cells were treated with FSMO and si-MRS in combination, and their effect on the interaction between overexpressed HSP90 and CDK4 was investigated.

Article Snippet: Primary antibodies for HA (mouse, sc-7392; Santa Cruz and rabbit, H6908; Sigma), Myc (mouse, sc-40; Santa Cruz), Flag (mouse, F3165; Sigma), Strep-HRP (2-1509-001; IBA), tubulin (mouse, T6074; Sigma), β-actin (mouse, A1978; Sigma), CDK4 (mouse, sc-23896 and rabbit, sc-260; Santa Cruz), cyclin D1 (rabbit, 04-221; Merckmillipore), p-Rb (rabbit, #3590; Cell Signaling), HSP90α/β (rabbit, sc-7947; Santa Cruz), CDC37 (rabbit, sc-5617; Santa Cruz), p16 (rabbit, sc-468; Santa Cruz), CDK3 (rabbit, sc-28256; Santa Cruz), CDK6 (mouse, sc-7961; Santa Cruz), and MRS (mouse, ab50793; Abcam) and a CDK antibody sampler kit (#9868; Cell Signaling) and a Cyclin antibody sampler kit (#9869; Cell Signaling) were used to perform immunoblotting, immunoprecipitation, and immunofluorescence staining.

Techniques: Transfection, Control, In Vitro, Pull Down Assay