Ab15954

Ubiquitin vinyl sulfone assays (UbVS) were performed by incubating human full‐length, chimera Ub‐R0RBR or human R0RBR, WT or mutant with an excess of UbVS (Boston Biochem) in 50 mM Tris–HCl pH 8, 150 mM NaCl, 1 mM TCEP for 30 min at 37°C. Reactions were stopped by the addition of 5× SDS–PAGE loading buffer with 100 mM dithiothreitol (DTT) and analyzed on SDS–PAGE gel stained with Coomassie blue or by western blotting. For western blotting, the samples on SDS–PAGE gel were transferred to Immune‐Blot PVDF membrane. The membrane was first blocked with 5% BSA in TBS‐T (0.05% Tween 20) overnight at 4°C and then incubated with 1:2,000 dilution of rabbit anti‐Parkin antibody (Ab15954, AbCam) in the blocking solution. The membrane was washed with TBS‐T and incubated with 1:10,000 dilution of horseradish peroxidase (HRP)‐coupled goat anti‐rabbit IgG antibodies (Cell Signaling) in TBS‐T. After washing in TBS‐T, chemiluminescence was detected using ECL Prime (Cytiva) and bands were visualized with film.

Primary antibodies were diluted using milk diluent/blocking solution concentrate (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland, USA). Secondary antibodies were diluted in 5% skim milk in 1× Tris-buffered saline Tween (TBST). Antibodies used in this study are: anti-HAP40 (a.a. 293-310, 1:3300; LTK BioLaboratories, Taiwan), anti-ADRM1 (ab157218, 1:1000; Abcam), anti-Mfn1 (ab57602, 1:1000; Abcam), anti-Mfn2 (GTX102058, 1:500; GeneTex), anti-OPA1 (sc-30572, 1:200; Santa Cruz Biotechnology, Inc.), anti-Fis1 (sc-98900, 1:200; Santa Cruz Biotechnology, Inc.), anti-Drp1 (8570, 1:1000; Cell signaling), anti-phospho Drp1(Ser616) (3455, 1:1000; Cell signaling), anti-Parkin (ab15954, 1:1000; Abcam), anti-PINK1 (ab75487, 1:500; Abcam), anti-PHB1 (2426, 1:1000; Cell signaling), anti-Tubulin (T6074, 1:5000; Sigma), anti-GAPDH (MAB374, 1:500; Millipore Bioscience Research Reagents) and anti-Actin (MAB1501, 1:1000; Millipore Bioscience Research Reagents).

For immunoprecipitation, the cells were washed twice, harvested in PBS, pelleted at 300 g for 5 min at 4 °C and then lysed in ice-cold IP lysis buffer containing 20 mM HEPES, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol pH 7.4, and protease inhibitor cocktail. Cell lysates were passed through a 23 G needle 20 times before incubating on ice for 30 min with shaking at 350 rpm. The insoluble fraction was removed by centrifuging at 14000 g for 10 min at 4 °C. The protein concentrations were estimated using DC Protein Assay reagent according to the manufacturer’s protocol. Lysates were incubated overnight on rotary shaker at 4 °C in Pierce spin columns with 5 µg of Ms anti-PARK2 (Abcam ab77924), Rb anti-PARK2 (Abcam, ab15954), Ms anti-BECN1 (Abcam, ab79937), Rb anti-FLAG (Sigma, F7425), Rb anti-BECN1 or Ms anti-MYC (Invitrogen, 46-0603) antibodies. The following morning 150 µl protein G-sepharose 4B beads (50% suspension in IP lysis buffer) were added and further incubated for 5 h on a rotary shaker at 4 °C. Beads were then washed 5 times with IP lysis buffer and immunoprecipitates were eluted in Laemmli buffer at 99 °C for 10 min. For immunoprecipitation experiments of mitochondrial and cytosolic fractions, the cells were fractionated in advance as described below.

Western blot was used to detect the protein levels.⁵⁹ (link) Protein samples were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus and then transferred to polyvinylidene fluoride (PVDF) nitrocellulose membranes (Millipore, USA), blocked with a mixture of 5% Skim Milk Powder and TBST (tris-buffered saline and Tween 20) at room temperature for 2 hr. Membranes were incubated with the primary antibodies at 4°C overnight and further incubated with the secondary antibodies for 2 hr at room temperature. Primary antibodies against IL-6 (ab208113), NLLRP3 (ab214185), Pro-IL-1β (ab106035), IL-1β p17 (ab106035), Pro-Casp1 (ab179515), Casp1 p10 (ab179515), p62 (ab109012), LC3 (ab51520), β-actin (ab8227), Parkin (ab15954), VDAC1 (ab15895), Tubulin (ab6046), GAPDH (ab9385), TRAF3 (ab36988), p65 (ab32536), and p-p65 (ab86299) were purchased from Abcam (Cambridge, UK), and secondary antibody was purchased from Baoshen (Beijing, China).