Ver 155008

The piriform cortices and the hippocampal formation were dissected from mouse brains at E17.5. The tissues were digested using 90 U of papain (Worthington Biochemical Corporation, Lakewood, NJ) in 1 ml of PBS(-) for 20 minutes at 37°C. The tissues were triturated and the dissociated cells were plated on polyethyleneimine-coated dishes in DMEM/10% fetal calf serum. After the cells attached to the dishes, the medium was changed to MACS Neuro Medium containing MACS NeuroBrew-21 (Miltenyl Biotec K. K., Tokyo, Japan), l-glutamine (2 mM), penicillin (100 U), and streptomycin (0.1 mg/ml). Hippocampal neurons were transfected with the pCAGGS FILIP IRES-GFP vector, the pCAGGS FILIP d872-1111 IRES-GFP vector, or the pCAGGS IRES-GFP vector and the pCAGGS tdTomato vector using Lipofectamine 2000 (Invitrogen) at day in vitro (DIV) 17. Piriform neurons were transfected with a pCAGGS tdTomato vector using Lipofectamine 2000 (Invitrogen) at DIV17. The cells were treated with an Hsc70/Hsp70 inhibitor (2 μM clofibric acid for 12 hours or 4 μM VER-155008 [Sigma-Aldrich] for 6 hours) and fixed using 4% PFA in 0.1 M phosphate buffer at DIV20. To calculate spine lengths, images were composed from Z-stacked images captured using a confocal microscope in ImageJ (National Institutes of Health, Bethesda, MD).

The recombinant C2I protein was purified and activated as described before60 (link). Recombinant C3bot was purified as described earlier61 (link). Ia and Ib were prepared as described earlier62 (link). Recombinant Hsp70, Hsc70 and Hsp90 proteins were purified as described63 (link)64 (link). Acridizinium derivatives 1–3 were prepared according to published protocols65 . Characterization of the compounds including NMR data can be found in the literature44 46 (link)65 . VER-155008 was purchased from Sigma-Aldrich.

All commercial chemicals and solvents were purchased from Sigma Aldrich or Fisher Scientific and used without further purification. The identity and purity of each product was characterized by MS, HPLC, TLC, and NMR. Purity of target compounds has been determined to be >95% by LC/MS on a Waters Autopurification system with PDA, MicroMass ZQ and ELSD detector and a reversed phase column (Waters X-Bridge C18, 4.6 × 150 mm, 5 µm) eluted with water/acetonitrile gradients, containing 0.1% TFA. Stock solutions of all inhibitors were prepared in molecular biology grade DMSO (Sigma Aldrich) at 1000× concentrations. The epiHSP70 ligands and probes, the epiHSP90 inhibitor PU-H71, the PU-beads and the control baits were generated using published protocols^{27 (link)–31 (link),80 (link)} and described in Supplementary Notes 1. Thymidine, nocodazole, RO-3306, MG132, paclitaxel, VER155008 and MKT077 were purchased from Sigma-Aldrich. Proteins were generated and purified using published protocols described in Supplementary Note 2^{81 (link)}.

FRET was visualized on an epifluorescence Carl Zeiss TM210 microscope using the three-filter method as described previously (24 (link)). The plasmid for YFP-HSP70 was generously provided by Harm Kampinga (University of Groningen, Netherlands). Cells were seeded on 29-mm glass bottom dishes and transfected with plasmids encoding YFP-HSP70 (0.2 μg) and wild type or mutant versions of CFP-SERT (0.8 μg). For experiments involving the HSP70 inhibitor VER-155008 (Sigma), the cells were incubated with the drug (40 μm) for 2 h prior to measuring FRET. The images were acquired using a 6× oil immersion objective and an automated filter wheel (Ludl Electronic Products, Hawthorne, NY) to allow for rapid switching between the fluorescence excitation and emission filters for CFP (I_CFP; excitation, 436 nm; emission, 480 nm; dichroic mirror, 455 nm), YFP (I_YFP; excitation, 500 nm; emission, 535 nm; dichroic mirror, 515 nm), and FRET (I_FRET; excitation, 436 nm; emission, 535 nm; dichroic mirror, 455 nm). The images were captured by a charge-coupled device camera and analyzed using the ImageJ PixFRET plug-in (25 (link)).

HEK293T cells were grown in DMEM (Sigma-Aldrich) plus 10% FBS (Sigma-Aldrich) and penicillin/streptomycin (P/S). Proteins were first separated on 10 or 12% polyacrylamide gels and then subjected to Westernblot transfer. The membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25 mM Tris-HCl pH 7.5, 150 mM NaCl). Primary antibodies used for Western blotting or ICC were anti-α-syn (BD, Cat# 610787), HRP coupled anti-Actin (Sigma, Cat# A3854), anti-HSP70 (AbCam, Cat#: ab47455), anti-GFP (Thermo Fischer, Cat#: MA5-15256), anti-Lamin-B1(AbCam, Cat#: ab16048), anti-pan cytokeratin (Thermo Fischer, Cat# MA5-13203) and anti-DNAJB6 (AbCam, cat# ab198995). Secondary antibodies used were anti-mouse HRP-conjugated antibodies (Dako), and RDye fluorescently labeled secondary antibodies (Li-Cor, UK). VER-155008 (Hsp70 inhibitor) was purchased from Sigma-Aldrich.

Mouse MA-10 Leydig cells and Chinese hamster ovary (CHO) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% fetal bovine serum and 20 mM HEPES. HEK293T cells were grown in DMEM supplemented with 2 mM glutamine, 10% fetal bovine serum (Thermo Scientific, Waltham, MA, USA), 100 units/mL penicillin, and 50 μg/mL streptomycin, and were maintained at 37 °C in a humidified incubator with 5% CO₂. Transient transfection in HEK293T and CHO cells was performed by using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Cells were plated onto 12-well plates 24 hrs before transfection. Various expression constructs were incubated with the transfection reagent for 20 min at room temperature, and DNA-lipofectamine diluted in Opti-MEM (Invitrogen, Carlsbad, CA, USA) was added to culture wells. After 6-h incubation at 37 °C, the medium was changed and the culture cells were maintained in the 37 °C incubator for 48 hrs. Where indicated, 17-AAG, 2-phenylethynesulfonamide (PES; also known as pifithrin-µ), and VER-155008 (Sigma, St. Louis, MO, USA), dissolved in 0.1% dimethyl sulfoxide (DMSO), was applied to the culture medium.