Irdye 800cw donkey anti rabbit igg

Mice were anesthetized with ketamine/xylazine and transcardially perfused with PBS. The brain and spinal cords were quickly harvested. The brain and spinal cords were dounced using a tissue douncer with RIPA buffer (pH 7.5, 25 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and the appropriate dilution of protease and phosphatase inhibitors. The dounced tissue was then sonicated and stored at −80 °C. Protein levels were normalized using a Bradford Assay and were assessed with Li-Cor Odyssey CLx infrared imaging system. Primary antibodies used for western blot were used as follows: goat anti-CCL21-unconjugated (1:100; AF457-SP; R&D Systems), rabbit anti-CCR7-unconjugated (1:100; ab32527; Abcam), rabbit anti-VEGFC-unconjugated (1:100; ab83905; Abcam), and chicken anti-ß-actin-unconjugated (1:1000; ab13822; Abcam). The appropriate secondary antibodies were used as follows: donkey anti-rabbit IgG-IRDye 800CW (1:2000; 926–32213; Li-Cor), donkey anti-goat IgG-IRDye 800CW (1:2000; 925–32214; Li-Cor), and donkey anti-chicken IgG-IRDye 680LT (1:2000; 926-68028; Li-Cor). For a detailed list of reagents, refer to Supplementary Table 1. Representative uncropped western blots for VEGFC and ß-actin can be found in Supplementary Fig. 11.

For total protein analysis, EVs were diluted to equal concentrations and run on polyacrylamide gels. Bands were visualized by silver staining (Thermo Fisher). For immunoblot analysis, EVs and control fractions were loaded onto a polyacrylamide gel and transferred onto a nitrocellulose membrane (GE). Membranes were blocked with 7% dry milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h. The primary antibodies used were diluted in 7% dry milk in TBST to the following final concentrations: GAPDH (Abcam 9485), 200 ng/ml; flotillin 2 (Cell Signaling 3244), 100 ng/ml; histone H3 (Abcam 1791), 200 ng/ml; SIVmac239 p27 monoclonal (4B2) (AIDS Reagent Database 2321), 1 µg/ml; SIV Nef monoclonal (clone 17.2) (AIDS Reagent Database 2659), 1 µg/ml; CD63 (H-193) (Santa Cruz Biotech sc-15363), 200 ng/ml; CD81 (Q-14) (Santa Cruz Biotech sc-31234), 200 ng/ml; GFP (ab290), 100 ng/ml. Secondary fluorescent antibodies were also diluted in 7% dry milk in TBST to the following final concentrations: donkey anti-rabbit IgG IRDye 800CW (LiCor P/N 926-32213), 100 ng/ml; donkey anti-goat IgG IRDye 680RD (P/N 926-68074), 100 ng/ml; donkey anti-mouse IgG IRDye 680RD (LiCor P/N 926-68072), 100 ng/ml. Peroxidase-labeled secondary antibodies were diluted in TBST to a final concentration of 100 ng/ml. Western blot assay images were obtained with the LiCor Odyssey system or X-ray film.

The rabbit phospho-site–specific GPR35 antiserum pSer³⁰⁰/pSer³⁰³-hGPR35a (Cat number (7TM0102C), raised against the sequence KAHKpSQDpSLCVTL, and the pSer²⁹⁸/pSer³⁰¹-mGPR35 antiserum (7TM0102B), raised against the sequence TPHKpSQDpSQILSLT, were developed in collaboration with 7TM Antibodies GmbH. IRDye 800CW donkey anti-rabbit IgG, IRDye 800CW donkey anti-goat IgG, and IRDye 800CW goat anti-rat IgG were from LI-COR Biosciences. Alexa Fluor 488-goat anti-rabbit IgG and Alexa Fluor 594-donkey anti-rat IgG were from Abcam. Mouse monoclonal anti-FLAG M2 was from Merck. Horseradish peroxidase anti-mouse (sheep) was from GE Healthcare. High affinity anti-HA (rat) and anti-HA affinity matrix were from Roche Diagnostics.

Protein samples (20 µg) were separated using 10% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane (PVDF, Merck Millipore, USA). Western blot was performed as previously described (Matsukawa et al., 2017). Primary antibodies used were anti-β-III-Tubulin (CST, Japan); anti-myelin basic protein (MBP) (Sigma, Japan); anti-glial fibrillary acidic protein (GFAP) (Sigma, Japan); anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:200, Santa Cruz, Japan); rabbit anti-Phospho-p53 (S15, CST, Japan) and rabbit anti-p53 (1:1000, CST, Japan). Secondary antibodies were IRDye 800CW donkey anti-rabbit IgG (1:1000) or IRDye 680LT goat anti-mouse (1:20000) from LI-COR, Inc., Lincoln, NE, USA. The signal was detected using the Odyssey FC Imaging System (LI-COR, Inc, NE, USA).

Phosphatase inhibitor cocktail IV (ab201115) and EDTA-free protease inhibitor cocktail (4693159001) were from Abcam (Cambridge, MA, USA) and Roche Diagnostics (Mannheim, Germany), respectively. [³H]-AMPA (DL-α-[5-methy-3H)) was obtained from Perkin Elmer (Boston, MA, USA; NET833250UC; 58.1 Ci/mmol). Potassium thiocyanate (KSCN; 207799), tetraethyl ammonium (TEA; T-2265) and latrunculin A (L5163) were procured from Sigma-Aldrich (St. Louis, MO, USA). Forskolin (HB1348) was from HelloBio. Rolipram (ab120029) and primary antibodies against PSD-95 (rabbit; ab18258) and GAPDH (mouse; ab8245) were from Abcam (Cambridge, MA, USA). Fluorescent secondary antibodies (IRDye^® 800 CW donkey anti-rabbit IgG and IRDye^® 680 RD goat anti-mouse IgG) were from LI-COR Biosciences (Lincoln, NE, USA). Other regents used in the study were of analytical grade and purchased either from Thermo Fisher Scientific or Sigma-Aldrich.