Anti mouse igg hrp linked whole antibody

PIF1 recombinant proteins were prepared using PIF1-his DNA (pET21a) provided by Enamul Huq (University of Texas at Austin, USA), and induced and purified using a commercial kit according to manufacturer’s instructions (Amersham). Polyclonal anti-PIF1 was obtained from rabbits immunized with PIF1 recombinant protein. PIF1 antibodies were further affinity-purified using PIF1 recombinant protein immobilized on nitrocellulose filters as described [61 (link)]. FR or R light irradiated WT and mutant seeds were harvested under green safety light at the time indicated in each experiment. 20 seeds were homogenized with homogenization buffer (0.0625 M Tris-HCl at pH 6.8, 1% [w/v] SDS, 10% [v/v] glycerol, 0.01% [v/v] 2-mercaptoethanol), and total proteins were separated by SDS-PAGE gel and transferred to a PVDF membrane (Amersham). PIF1 and UGPase proteins were detected using 1:500 dilution of anti-PIF1 or 1:10,000 dilution of anti-UGPase (Agrisera), and anti-rabbit IgG HRP-linked whole antibody (GE healthcare) in a 1:10,000 dilution was used as a secondary antibody. PHYA proteins were detected using 1:5,000 dilution of anti-phyA (kindly provided by Akira Nagatani; Kyoto University, Japan) and the anti-mouse IgG HRP-linked whole antibody (GE healthcare) in a 1:10,000 dilution was used as a secondary antibody [17 (link)]. Quantification of band intensity was performed using imageJ.

With regard to the primary antibodies for Western blotting, anti-CD11b antibody (1:500, Assay Biotech), anti-CD16-B (Z64) mouse monoclonal IgG2b (1:200, Santa Cruz Biotechnology), anti-GAPDH (14C10) rabbit mAb (1:10000, Cell Signaling Technology), and anti-β-actin antibody (1:3000, Cell Signaling Technology) were used.
Anti-NAIP antiserum (1:5000, Neugen Pharma Inc.), which is commercially available (Wako, product code 019-24251), was raised by immunizing Japanese White rabbit with full-length NAIP synthesized using cell-free protein synthesis (WEPRO1240G Expression Kit) according to the manufacturer’s instruction^{24 (link)}, and was used as the primary antibody for both Western blotting and dot blotting.
Anti-rabbit IgG, horseradish peroxidase (HRP)-linked whole antibody (1:10000, GE Healthcare) and anti-mouse IgG, HRP-linked whole antibody (1:10000, GE Healthcare) were used as secondary antibodies.

Lysates of sEVs were extracted by M-PER (Thermo Fisher Scientific). Total proteins were separated by SDS-PAGE using 4–20% Novex 4–20% Tris-Glycine Mini Gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Thermo Fisher Scientific). Electrophoresis, blotting, and antibody treatment were performed using a Mini Gel Tank (Thermo Fisher Scientific), Pierce Power Blotter Stainer System (Thermo Fisher Scientific), and iBind Western Systems following the manufacturer’s instructions. Primary antibodies (Exosome-anti CD9; 10626D; dilution: 1:50, CD63; 10628D; dilution: 1:50, and CD81; 10630D; dilution: 1:50 for western blot) were purchased from Thermo Fisher Scientific and secondary antibodies (anti-mouse IgG HRP-linked whole antibody) were purchased from GE Healthcare (Chicago, IL, USA) (Supplementary Fig. 1) All blots derive from the same experiment and were processed in parallel.