Anti parkin prk8

For affinity enrichment of Parkin, 5 µM (2.5 µg) of the respective Strep-Ub / NEDD8 variant was incubated with 10 µM Parkin (wild-type, ∆Ubl or H302A) for 30 min on ice. The mixture was added to 10 µl Strep-Tactin XT 4Flow beads (iba), preequilibrated with two times 200 µl binding buffer (1x PBS, 2 mM MgCl₂, 1 mM DTT). Upon incubation at RT for 3 h in an overhead shaker, the beads were washed three times with 200 µl lysis buffer each. Bound proteins were eluted by incubating the beads for 5 min on ice with two times 30 µl elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin). The eluates were combined and 25% of them were applied to SDS-PAGE followed by Western blot analysis using an antibody specific for Parkin (anti-Parkin PRK8, 1:1,000 (#sc-32282, Santa Cruz)) and as secondary antibody anti-mouse HRP (1:15,000; #115-035-062, Jackson ImmunoResearch).

Cells were seeded at 6 × 10⁴ in 24-well plates with cover glass for overnight culture and infected with GAS for 30 min. Extracellular bacteria were killed by the use of 100 μg/ml gentamicin. At various time points postinfection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-LC3 (pM036; MBL), anti-Gal-3 (M3/38; Santa Cruz), anti-Gal-8 (D-18; Santa Cruz), anti-ubiquitin (ab7780; Abcam, Inc.), and anti-parkin (PRK8; Santa Cruz) antibodies at room temperature for 1 h. After the cells were washed with PBS, they were stained with Alexa Fluor-conjugated secondary antibodies and DAPI (4′,6-diamidino-2-phenylindole) for 1 h, and the samples were then analyzed by confocal microscopy (FV1000; Olympus).

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Hyclone) supplemented with 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (Hyclone). Predesigned siRNA duplexes were purchased from Bioneer, and 10 nM siRNA transfection was carried out using RNAiMAX (Invitrogen, Waltham, MA, USA). Cells and tissues were lysed with N-PER™ and T-PER™ lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitor cocktail (Roche, Hoffmann-La Roche AG, Basel, Switzerland), respectively. Lysates were resolved by SDS-PAGE gels and transferred onto nitrocellulose (NC) membranes (Merck Millipore, Burlington, MA, USA), and then antibodies were detected using the D-Plus ECL Femto System (Dongin Bio, Seoul, Korea) and the ImageQuant LAS4000 (GE Healthcare, Chicago, IL, USA). Antibodies used for immunoblotting included the following: anti-Ubiquitin (P4D1, Santa Cruz, Santa Cruz Biotechnology, Dallas, TX, USA), anti-UBE2H (18-Z, Santa Cruz), anti-Parkin (PRK8, Santa Cruz), anti-GAPDH (0411, Santa Cruz), anti-β-actin (C4, Santa Cruz), anti-Tau (Abcam, Cambridge, UK), and anti-UBE2L6 (MyBioSource, San Diego, CA, USA).