Anti myc

To check the expression or positioning of tagged proteins, yeast cells were cultured in YPG medium, collected at OD 10.0 and analysed by western blot according to the method described by Sui et al.58 (link). The cytoplasmic and mitochondrial fractions of engineered strains were separated using a mitochondria extraction kit (Bioversion). Total protein in full cells were extracted using a yeast protein extraction kit (Sangon Biotech, China). For comparison of the protein expression in cytoplasm and mitochondria engineering strains, total protein in full cells and each subcellular fraction were measured using a BCA protein assay kit (Sangon Biotech, China) and 4 μg of protein was loaded in each lane. The protein samples were fractionated on 12% SDS–polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated with the indicated primary and secondary antibodies (anti-myc, Clontech; anti-porin, Invitrogen; anti-flag, MBL). Proteins were ultimately visualized by enhanced chemiluminescence and autoradiography (ECL; Thermo Scientific, Waltham, MA, UK).

Cells were transformed with a plasmid (YEp352-6HisUb) harbouring 6His-tagged ubiquitin under the CUP1-inducible promoter51 (link). The cells were grown in media lacking uracil (to select for the plasmid). 6His-Ubiquitin was induced by 24 h treatment with 0.1 mM CuSO₄. Cells were harvested and lysed with guanidinium lysis buffer (6 M guanidine hydrochloride, 100 mM sodium phosphate buffer pH8.0, 10 mM Tris-HCl pH8.0, 10 mM imidazole, 10 mM β-mercaptoethanol, 0.1% Triton X-100, 2.5 mg ml⁻¹ N-methyl maleimide, 0.1 mM MG-132, 1 × protease inhibitor). Purification of 6His-ubiquitinated proteins was performed using the Ni-NTA (Ni²⁺-nitrilotriacetic acid) agarose beads (QIAGEN). The beads were washed with urea buffer (8 M urea, 100 mM sodium phosphate buffer pH 6.4, Tris-HCl pH 6.4, 10 mM imidazole, 10 mM β-mercaptoethanol, 0.1% Triton X-100) and subsequently eluted with protein sample buffer. Eluted protein samples were separated by SDS–PAGE and analysed by western blots using anti-Myc (Clontech) and anti-His antibodies (Novagen).

Positions reflect the nucleotide location in the NCBI Reference Sequences: NP_000542.1 (VHL) and NM_058195.3 (p14ARF). Primer nomenclature, positions, directions and sequences were set as described in Table 1. Protein extracts were obtained as in31 , subjected to SDS-Page (NuPAGE- Invitrogen®) and probed with anti-MYC (CLONTECH, 1:5000) and anti-HA (SIGMA, 1:5000) antibodies. The expression level of the fusion proteins was then quantified with the Image J software (available at http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health).

Antibodies used for immunoblot analysis were the following: anti-NLRP3, anti-IL-1β, anti-caspase-1, anti-caspase-9 and anti-cytochrome c (Cell Signaling); and anti-Myc (Clontech Laboratories), anti-Flag (Sigma-Aldrich), anti-HA (Cell Signaling), anti-ASC (Santa Cruz Biotechnology), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich), goat anti-rabbit HRP-linked antibody, and horse anti-mouse HRP-linked antibody (Cell Signaling). Purified cytochrome c protein (Sigma-Aldrich), Profect-P1-lipid based protein delivery reagent (Targeting Systems), cardiolipin beads (Echelon), and the mitochondrial fractionation kit (Active Motif) were purchased and used as directed by the manufactures. The LPS, ATP, and Poly (dA-dT) were purchased from Sigma-Aldrich.

HepG2 cells transfected with pCMVMycPod-1 were fixed with 1% formaldehyde for 10 min. ChIP assays were performed using the ChIP-IT Express kit (Active Motif, Rixensart, Belgium) following the manufacturer's instructions. Chromatin was fragmented by sonication with eight 10-s pulses at 25 μm amplitude in a VCX130PB ultrasonic processor (Sonics & Materials, CT, USA). Most resulting chromatin fragments ranged from 200 bp to 600 bp. Sheared chromatin was incubated with 3 μg anti-MYC (Clontech) or with 1 μg IgG as negative control (ChIP-IT Control Kit-Human, Active Motif).

For immunocytochemistry, primary cortical neurons were seeded on poly-l-lysine-coated cover slips placed in Petri dishes and infected with LV-GFP, LV-GFP-α-Syn, LV-RFP, or LV-RFP-PINK1, or co-infected with LV-GFP-α-Syn and LV-RFP-PINK1 for 3 days. Neurons were then fixed with 3.7% paraformaldehyde for 20 min, washed in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. Fixed cells were incubated with an anti-tubulin III antibody (Abcam) overnight at 4°C, followed by incubation with Alexa Fluor 647-labeled secondary antibody for 1 h at room temperature. Cell nuclei were visualized by counterstaining with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). Additionally, cultured SK-N-SH cells were transfected with myc/α-Syn or flag/PINK1, or co-transfected with myc/α-Syn and flag/PINK1 plasmids for 24 h. Cells were then fixed with 3.7% paraformaldehyde for 20 min, washed in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. After overnight incubation at 4°C with an anti-myc (Clontech, Mountain View, CA, USA) or anti-flag (ProteinTech) antibody, cells were labeled with Alexa Fluor 488- or 594-conjugated secondary antibody (1:500) for 1 h at room temperature. Nuclei were visualized by counterstaining with DAPI.