Anti mouse igg hrp

Polyclonal anti-TOM1L1 (1: 2,000) antibodies were as described in ref. 23 (link). The monoclonal anti-TOM1L1 antibody was from Covalab (Lyon, France). Antibodies against ERBB2 (1:1,000), p-CortactinY421 (1:500), p-AKTS473 (1:1,000), AKT (1:1,000), P44/P42 MAPK (1:1,000) and p-P44/P42 MAPK (1:1,000) were from Cell Signaling Technology (Danvers, USA). Antibody against Cortactin (1:500) was from Millipore (Billeria, USA) and antibodies against MT1-MMP (1:100 for if or 1:500 for immunoblotting) and TOLLIP (1:200) from Abcam (Cambridge, UK). Anti-tubulin (1:2,000), HA-tag (1:4,000) and p-Tyr 4G10 (1:50) antibodies were from N. Morin, C. Gauthier-Rouvière and P. Mangeat, respectively (CRBM, Montpellier, France). Anti-rabbit IgG-HRP (1:5,000) and anti-mouse IgG-HRP (1:5,000) were from GE Healthcare (Fairfield, USA). Anti-rabbit and anti-mouse IgG coupled to Alexa-Fluor 488, Alexa-Fluor 594 and Alexa-Fluor 405 (1:1,000) were from Life Technologies (Carlsbad, USA). Alexa-Fluor 594-Phalloidin (Life Technologies) was used to visualize F-actin. GFP-Nanotrap technology antibodies were from Chromotek (Planegg-Martinsried, Germany).

Antibodies against phospho-SHIP1 (Tyr1020), phospho-SHP-1 (Tyr564) (D11G5), phospho-Src family (Tyr416) (D49G4), phospho-p38 MAPK (Thr180/Tyr182) (3D7), phospho-(Ser) PKC substrate, phospho-p40^phox (Thr154), and Rac1/2/3 antibodies were purchased from Cell Signaling Technology. Phospho-ERK (E-4), p38α/β MAPK (H-147), p47^phox (H-195), p22^phox (C-17), Rab5 (D-11), Rab7 (H-50), calnexin (H-70), c-Myc (9E10), and COX-2 (C-20) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The iNOS/NOS type II (54/iNOS) antibody was purchased from BD Biosciences, and the β-actin (AC-15) antibody was purchased from Sigma-Aldrich. Secondary antibodies for Western blotting, ECL™ anti-rabbit, and anti-mouse IgG HRP were obtained from GE Healthcare. For immunofluorescence and confocal laser microscopy, Alexa Fluor^®488 goat anti-mouse IgG, Alexa Fluor^®488 goat anti-rabbit IgG, Alexa Fluor^®488 donkey anti-goat IgG, Alexa Fluor^®594 goat anti-rabbit IgG, and Alexa Fluor^®647 donkey anti-mouse IgG were obtained from Invitrogen. Phorbol 12-myristate 13-acetate (PMA) and pure lipopolysaccharide (LPS) from E. coli O111:B4 (L3024) were purchased from Sigma-Aldrich. The inhibitors, bisindolylmaleimide I (BIM-1), SB203580, and LY294992 were obtained from Calbiochem (San Diego, CA, USA).

Samples were separated on Native-PAGE^™ Bis-Tris Gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% BSA in TBST (Tris-buffered Saline-Tween 20) overnight at 4 °C. Then the membranes were incubated with purified MBP-PFFNB2-FLAG (~4 μg/ml) or mouse anti-α-syn mAb (1:2000 dilution, BD Biosciences, Cat no. 610787) in TBST with 5% Bovine Serum Albumin overnight at 4 °C. Mouse-anti-Flag-HRP antibody (1:5000 dilution, Sigma-Aldrich, Cat no. A8592) or anti-mouse IgG-HRP (1:5000 dilution, GE Healthcare, Cat no. NA931) were used as secondary antibody followed by incubation with SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific). The images were acquired and processed with ImageQuant LAS 4000mini scanner (GE Healthcare Life Sciences) and Amersham Image 600 (GE Healthcare Life Sciences).
The soluble and insoluble fractions of brain lysates were resolved on 15% Tris-glycine gel and transferred to PVDF membranes for analysis with rabbit anti-α-syn (1:1000 dilution, Cell signaling, Cat#4179), rabbit anti-α-syn (pS129) (dilution 1:1000, Abcam, Cat no. ab51253) and mouse anti-ß-actin peroxidase (1:10,000 dilution, Millipore Sigma, Cat no. A3854) antibodies.

anti-p42/44 MAPKs (#4695S), anti-p42/44 MAPKs pT202/Y204 (#4370S), anti-p38 MAPK (#9212), anti-p38 MAPK pT180/Y182 (#9211S), anti-AKT (#9272S), anti-AKT pS473 (#4060S), anti-EPHA2 pY594 (#3970S), anti-Src pY419 (#2101L), anti-Stat3 pY705(#9131S) (CST), anti-EPHA2 (#6997S), anti-Stat3 (#9139S), anti-Myc (#2276S) and anti-pTyr clone pY1000 Sepharose bead conjugated (PTM Scan) were from CST, anti-Src specific (2.17) antibody (a gift from Dr S Parsons, University of Virginia, VA, USA), anti-FLAG (M2 antibody, Sigma Aldrich), and anti-GFP (Chromotek), anti-tubulin (gift from N. Morin, CRBM, Montpellier, France), anti-pTyr 4G10 (gift from P. Mangeat, CRBM, Montpellier, France), anti-cst1 (that recognizes Src, Fyn and Yes) was described in [39 (link)]. Anti-rabbit IgG-HRP and anti-mouse IgG-HRP (GE Healthcare). Anti-Mib1h (Dako)(KI67), anti-active-caspase3 (AP175; CST), anti-CD31 (Ab28364, Abcam).

Western blotting was performed by standard methods. The following antibodies were used: 1/3000 rabbit anti‐YAP/TAZ (Cell Signaling Technology, Danvers, MA, USA; #8418), 1/10 000 mouse anti‐GAPDH (EMD Millipore, Burlington, MA, USA; MAB374), 1/3000 rabbit PAPR (Cell Signaling Technology; #9542), 1/5000 mouse anti‐E2F1 (Proteintech, Rosemont, IL, USA; 66515‐1‐Ig), 1/2000 rabbit anti‐FANCD2 (Proteintech; 204006‐1‐AP), 1/3000 mouse anti‐epidermal growth factor receptor (EGFR; Cell Signaling, #2239), 1/5000 anti‐mouse IgG‐HRP (GE Healthcare, Buckinghamshire, UK) and 1/5000 anti‐rabbit IgG‐HRP (GE Healthcare). All antibodies were diluted with Can Get Signal reagent (TOYOBO, Osaka, Japan). The band intensity was quantified by imagej (National Institute of Health, Bethesda, MD, USA), and the percentage of cleaved PARP was calculated as the percentages of cleaved PARP in total PARP: cleaved PARP/(full‐length PARP + cleaved PARP) × 100 in each sample.

H3K27ac (39,133, Active Motif Lot # 31814008), H3K27ac (39,685, Active Motif, no. 14517014), H3K18ac (ab1191, Abcam, no. GR3211480–1), H3K27me3 (9756, Cell Signaling), H3K9ac (ab4441, Abcam), H4K16ac (39,167, Active Motif), H3 general (ab1791, Abcam, no. GR177884–2), Spike-In antibody (61,686, Active Motif, Lot# 00419007), p300 (sc-584, Santa Cruz, Lot # F3016), Gapdh (2118, Cell Signaling, Lot # 10), CBP(D6C5) (7389S, Cell Signaling), p53(CM5) (NCL-L-p53-CM5p, Leica Biosystems), PKCs p2056 (ab18192, Abcam), KAP1/TRIM29 pS824 (A300-767A, Bethyl), Acetyl-p53 (Lys379) (2570, Cell Signaling), anti-mouse IgG-HRP (NA93V, GE, no.9773218), anti-rabbit IgG-HRP (170–6515, Bio-Rad, no. 350003248).