Peroxidase affinipure donkey anti rabbit igg

For western blots, cell pellets were lysed in whole‐cell extraction buffer (150 mM NaCl, 50 mM Tris–pH 7.5, 1% NP‐40 supplemented with proteinase and phosphatase inhibitors) for 20 min at 4°C while vigorously shaking, insoluble material was spun out, relative protein concentration was determined by BCA (BioRad). SDS‐sample buffer with 100 mM DTT was added to samples, which were loaded onto 8% Protein gels and separated by electrophoresis at 90–140 V for 2–3 h. Proteins were transferred and immobilized onto a nitrocellulose membrane (GE Healthcare) by electrophoresis for 2 h at RT in a standard transfer buffer containing 20% methanol. Membranes were blocked in 5% nonfat dry milk in TBS‐T. All rabbit primary antibodies were probed with Peroxidase AffiniPure Donkey anti‐rabbit IgG (Jackson ImmunoResearch) and all mouse primary antibodies were probed with Peroxidase AffiniPure Donkey anti‐mouse IgG (Jackson ImmunoResearch). Chemiluminescent signals were visualized with Fusion Solo Imager.

The following primary antibodies were used for western blot analysis: rabbit anti-ATG9A (Abcam, ab108338, 1:1000), mouse anti-GAPDH (Meridian Life Sciences, H86504M, 1:750,000), mouse IgG2b anti-PINK1 (Novus, NBP2-36488, 1:10,000), rabbit anti-phospho S65 ubiquitin (Cell Signaling Technologies, 62802, 1:20,000), rabbit anti-PGAM5 (Abcam, AB126534, 1:15,000). The following secondary antibodies from Jackson Immunoresearch were used for western blot analysis: Peroxidase-AffiniPure donkey anti-mouse IgG (H+L) (715-035-150), Peroxidase-AffiniPure donkey anti-rabbit IgG (711-035-152), Peroxidase-AffiniPure goat anti-mouse IgG Fcγ subclass 2b specific (115-035-207). The following primary antibodies were used for immunofluorescence analysis: rabbit anti-ATG9A (Abcam, ab108338, 1:200), mouse IgG1 anti-HSP60 (PTG, 66041-1-Ig, 1:4000). The following secondary antibodies were used for immunofluorescence analysis: goat anti-rabbit AlexaFluor 568 (Molecular Probes, A11011, 1:1000), goat anti-mouse AlexaFluor 647 (Molecular Probes, A21235, 1:1000).

About 20 fly hearts per condition were lysed with 20 mM Tris-HCl (pH 8.0), 100 mM NaCl buffer supplied with protease inhibitor at 4°C for 20 min. After centrifugation at 14,000 rpm for 30 min, the supernatants were denatured with Laemmli sample buffer at 95°C for 5 min. Proteins were separated by Mini-PROTEAN^® TGX Precast Gels (Bio-Rad Laboratories, Inc.), transferred to PVDF membranes, immunoblotted with the following primary and secondary antibodies, and visualized with Pierce ECL Western Blotting Substrate. The antibodies used in western blot were: anti-phospho-Akt (Ser473) (Cell signaling technology #4060, 1:1000), anti-Akt (pan) (Cell signaling technology #4691, 1:2000), anti-beta-actin (Cell signaling technology #4967, 1:2000), Peroxidase AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch # 711-035-152, 1:5000).

Resuspending HDF cell pellets were prepared for protein lysates with SDS sample buffer (50 mM Tris-HCl 2% SDS, 0.1% Bromophenol blue, 10% glycerol). Protein quantification was performed with an RC DC protein assay kit (Bio-Rad, Hercules, CA, USA). 15 μg of protein in each sample was separated in 10% Tris-glycine gel and transferred to a nitrocellulose membrane. Membranes were blocked with TBST buffer (20 mM Tris, 150 mM NaCl, 0.5% Tween-20) supplemented with 5% BSA and incubated with primary antibody. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies. Similarly, after washing with TBST, an enhanced chemiluminescence solution (Thermo Scientific, Waltham, MA, USA) was added for detection. The primary antibodies are: Rabbit anti-AMPK (2534; Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Rabbit anti-pAMPK (2535; Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), Rabbit anti-β actin (8457; Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution). The secondary antibodies are: Peroxidase AffiniPure Donkey Anti-Rabbit IgG (711-035-152; Jackson Immunoresearch, West Grove, PA, USA, 1:5000 dilution).

Protein extraction from SCG neurons, PC12 cells and mouse SN tissues was performed using lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100), followed by low-intensity sonication pulses. The above protein extracts or HPLC-purified αSyn after the in vitro reaction were separated by 12.5% or 15% acrylamide gels, respectively, followed by transfer to PVDF membranes or Coomassie brilliant blue staining. Blots were incubated with blocking buffer (5% skim milk in TBS-T) and immunostained overnight at 4 °C with the following primary antibodies: mouse anti-αSyn (1:1000, BD Transduction Laboratories, 610787), rabbit anti-TH (1:1000), mouse anti-RFP (1:2000, MBL, M208-3), mouse anti-βIII-tubulin (Tuj1, 1:2000, R&D Systems, MAB1195) antibodies and mouse Y136DOPAmab. After washing with TBS-T, the membranes were incubated with Peroxidase AffiniPure Donkey Anti-Mouse IgG (1:3000) or Peroxidase AffiniPure Donkey Anti-Rabbit IgG (1:3000, Jackson ImmunoResearch, 711-035-152) for 1 h at RT. After washing, the immunoblots were developed using ECL Prime detection reagent (Cytiva). Chemiluminescence was detected by Fusion solo S (VILBER). The uncropped blots images are provided in the Source Data file.
For the antibody absorption assay, Y136DOPAmab was preincubated with 1 µg/mL 136DOPA or 136Y peptides overnight at 4 °C before use.

Following antibodies were used: mouse anti beta-Actin (Abcam, ab6276, 1:1000), mouse anti-H2A.X phospho S139 (Millipore, 05-636, 1:1000), mouse anti-Histone H3 phospho-S10 (H3-pS10; Abcam, ab5176, 1:1000), rabbit anti-cleaved PARP (Cell Signaling, #9541, 1:1000), rabbit anti-PDL1 (Cell Signaling, #13684, 1:1000), rabbit anti-pTBK1 (Cell Signaling, #5483, 1:500), rabbit anti-TBK1 (Cell Signaling, #3504, 1:1000), rabbit anti-pSTING (Cell Signaling, #50907, 1:500), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-cGAS (Cell Signaling, #15102, 1:1000), rabbit anti-MTH1 (Novus Biologicals, NB100-109, 1:1000). Secondary antibodies were: Peroxidase AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch, 711-035-152, 1:5000), Peroxidase AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch, 715-035-150, 1:5000), IRDye 680RD Goat Anti-Mouse IgG (LI-COR, 926-68072, 1:5000) and IRDye 800CW Donkey Anti-Rabbit IgG (LI-COR, 926-32213, 1:5000).