Pyr 41

Infection experiments of iDCs or mDCs were performed as recently described [82 (link),83 (link)]. Briefly, iDCs or mDCs were harvested and washed in RPMI 1640. Cells (1 × 10⁶–2 × 10⁶) were resuspended in 300–350 µL infection medium (RPMI 1640 supplemented with 20 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; Lonza)) containing a defined amount of HSV-1 or HSV-2 plaque forming units (pfu). For HSV-1 infection, a multiplicity of infection (MOI) of 1 or 2 was used (as indicated), whereas HSV-2 infection experiments were performed using an MOI of 5. As a mock control, cells were treated with infection medium supplemented with the respective amount of MNT buffer (30 mM MES, 100 mM NaCl, and 20 mM Tris). After 1 h of infection at 37 °C and shaking at 300 rpm cells were collected via centrifugation. Subsequently, cells were cultured in DC medium supplemented with 40 U/mL GM-CSF and 250 U/mL IL-4. To block proteasomal degradation either 10 µM MG-132 (Enzo Life Science, Lörrach, Germany) or 2 µM bortezomib (BZ; Santa Cruz Biotechnology, Heidelberg, Germany) were added 1 hpi (hour post infection). For blocking the ubiquitin E1-activating enzyme, 80 µM PYR-41 (Sigma-Aldrich) was added 4 hpi. As solvent control, dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to the medium.

Antibodies against β-actin (ab8226; RRID:AB_306371), human Aβ40 (ab12265; RRID:AB_298985), human Aβ42 (ab12267; RRID:AB_298987), LRP (ab92544; RRID:AB_2234877), RAGE (ab3611; RRID:AB_303947), APP (ab11118; RRID:AB_442855) and 20S proteasome (ab109530; RRID:AB_10860339; antibody raised against a synthetic peptide within the human proteasome 20S C2 unit (amino acids: 250–350; C terminal)), as well as cyclosporine A (CSA; ab120114) were purchased from Abcam (Cambridge, MA, USA). Modified Dulbecco’s phosphate buffered saline (DPBS; with 0.9 mM Ca²⁺ and 0.5 mM Mg²⁺) was purchased from HyClone (Logan, UT, USA). Complete™ protease inhibitor was purchased from Roche (Mannheim, Germany). C219 antibody against P-gp was purchased from ThermoFisher (MA126528; RRID:AB_795165; Waltham, MA, USA). Fluorescein-hAβ₄₂ [fluorescein-Aβ_(1–42)] was purchased from rPeptide (Bogart, GA, USA). [N- (4-nitrobenzofurazan-7-yl)-D-Lys8]-cyclosporine A (NBD-CSA) was custom-synthesized by R. Wenger (Basel, Switzerland; Wenger, 1986 (link)). PSC833 was a kind gift from Novartis (Basel, Switzerland). PYR41, CelLytic™ M, Ficoll^® PM 400, bovine serum albumin and all other chemicals were purchased at the highest grade from Sigma-Aldrich (St. Louis, MO, USA).

K562 cells, the human pre-B cell line Nalm-6, and the TOP2B−/− derivatives of Nalm-6 were grown in RPMI medium containing 10% FBS and 5% penicillin-streptomycin (%v/v) and incubated at 37°C, 5% CO₂. MLN7243 [TAK-243, sulfamic acid, ((1R,2R,3S,4R)-2,3-dihydroxy-4-((2-(3-((trifluoromethyl)thio)phenyl)pyrazolo(1,5-a)pyrimidin-7-yl)amino)cyclopentyl)methyl ester] (Hyer et al., 2018 (link)) was purchased from Active Biochem (Hong Kong). PRT4165 (Ismail et al., 2013 (link)) (2-pyridin-3-ylmethylene-indan-1,3-dione) was purchased from Merck Millipore, MA. UAE1 and UBA6 siRNA were purchased from ThermoFisher Scientific (MA, siRNA ID s599 and s30515, respectively). Etoposide and MG132 were purchased from Sigma-Aldrich (Dorset, UK) as was PYR41 ( 4[4-(5-Nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester).

Doxorubicin, puromycin, cycloheximide, leptomycin B, PYR-41, α-amanitin, actinomycin D, 2-D08 (all Sigma, Budapest, Hungary), 17-AAG (Reagents Direct, Encinitas, USA), MLN4924 (EMD Millipore, Darmstadt, Germany) and MLN7243 (Chemgood, Glen Allen, USA) were diluted to the final concentrations indicated in the Figures and added to live cells in complete DMEM medium for the time indicated, prior to fixing and lysis.

Beclin-1 (mouse, 2A4; rabbit, D40C5), GFP (rabbit, D5.1), LC3b (E5Q2K), Rab5 (E6N8S), and cleaved caspase-3 Abs were from Cell Signaling Technology. EEA-1 and full-length caspase-3 Abs were from BD Transduction Laboratory. LAMP2 (H4B4), GAPDH (FL-335), GFP (mouse, B-2), c-Myc (9E10), Bcl-2 (C-2), and horseradish peroxidase (HRP)-conjugated secondary Abs (αmouse, αrabbit, αrat, and αgoat IgGs) were from Santa Cruz Biotechnology. Mono- and polyubiquitinylated conjugated antibodies (FK2) (αUb) were obtained from Enzo Life Sciences. The p62/SQSTM1 Ab, chloroquine, 3-methyladenine, rapamycin, PYR-41 (ubiquitin-activating enzyme E1 inhibitor), proteinase K, and lysozyme (from hen egg white) were purchased from Sigma. ProLong Gold antifade mounting medium with DAPI (4′,6-diamidino-2-phenylindole), paraformaldehyde (PFA), Halt protease and phosphatase inhibitor cocktail, Dynabeads (M-270 Epoxy and A/G-agarose), dithiobis(succinimidyl propionate) (DSP), and Alexa 488/594-conjugated secondary Abs were purchased from Thermo Fisher Scientific. Recombinant phosphatidylinositol 3-kinase p110δ/p85α, phosphatidylinositol 4-kinase type 2 alpha (PI4K-2α), phosphatidylserine, and all utilized phosphoinositides were purchased from Echelon Biosciences. The PI3K inhibitors, wortmannin and LY294002 were obtained from Calbiochem and dissolved in the diluent dimethyl sulfoxide (DMSO; Sigma).