Peroxidase affinipure donkey anti mouse igg

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.

Liver tissues were collected at the indicated stages. All samples were lysed in RIPA lysis buffer (Beyotime, P0013B) containing protease inhibitors (Roche, 11836153001) for 30 min on ice, and then centrifuged at 13,500g for 5 min to collect the supernatant. All samples were mixed with loading buffer (Beyotime, p0015L) and boiled for 10 min. Because the molecular weights between FAH (46 kDa) and GAPDH (36 kDa) are close, we loaded the same amount of protein into two gels to detect FAH and GAPDG, respectively. Western blot analyses were performed with precast gradient gels (Beyotime, P0469M) and transferred onto polyvinyli denefluoride membranes (Millipore, IPVH00010). After blocking in PBST containing 5% BSA, the membranes were incubated with primary antibodies overnight at 4 °C, then washed three times and incubated with HRP-conjugated secondary antibodies. Signals were detected by incubating with chemiluminescent HRP substrate (Thermo Fisher Scientific, WBKLS0500). The following antibodies were used: FAH (Abclonal, A13492; 1:500), GAPDH (Proteintech, 60004-1-IG; 1:2,000), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-047; 1:4,000) and Peroxidase AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch, 715-035-150; 1:4,000).

Primary antibodies for immunoblotting including dot blot analysis and western blot analysis were performed with antibodies against Amyloid Fibril-Conformation-Specific (mOC87, abcam, Cat#: ab201062, 1:8000), SNAP25 (Synaptic systems, Cat#: 111-002, 1:2000), VAMP2 (abcam, Cat#: ab3347, 1:1000), β-actin (4D3, Bioworld Technology, Cat#: BS6007M, 1:5000). Secondary antibodies were conjugated with peroxidase affinipure donkey anti-rabbit IgG (H + L) (#711-035-152, Jackson ImmunoResearch, 1:10,000), Peroxidase affinipure donkey anti-mouse IgG (H + L) (#715-035-151, Jackson ImmunoResearch, 1:10,000).
Primary antibodies for immunohistochemistry were directed against purified anti-β-Amyliod 1-16 (6E10, Covance, SIG-39320, 1:500); MAP2 (AP20, Millipore, Cat#: MAB3418, 1:1,000) and SNAP25 (Synaptic systems, Cat#: 111-002, 1:2,000). Secondary antibodies were conjugated with Cy^TM3 affinipure donkey anti-mouse IgG (H + L) (#715-165-151, 1:400), Alexa Fluor® 488 affinipure donkey anti-rabbit IgG (H + L) (#711-545-152, 1:400).

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).

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.