Goat pab to mouse igg

The coding sequence of SRL2 was amplified by PCR and then subcloned into vector pJIT163-GFP to obtain the SRL2–GFP construct. The resultant construct was introduced into rice protoplasts and onion epidermal cells. Transfection of rice protoplasts with SRL–GFP was performed as described by Li et al. (2009) (link). For the transformation of onion cells, those plasmids were bombarded into onion epidermal cells using a PDS-1000 ⁄ He particle gun (Bio-Rad). The green fluorescence was observed by confocal laser-scanning microscopy (Zeiss LSM 510 META) with an argon laser excitation wavelength of 488nm.
Onion epidermis was ground in liquid nitrogen. Total protein was extracted with protein extraction buffer (50mM TRIS–HCl at pH 7.5, 150mM NaCl, 5mM EDTA, 0.2% NP-40, 0.1% Triton X-100, and Complete protease inhibitor cocktail, Roche), usually 600 μl for 0.2g onion epidermis. The extracts were centrifuged at 16 000rpm for 15min at 4 ℃, and the supernatants were collected for Western blot analysis. Western blot analysis was performed with a monoclonal mouse anti-GFP (Abmart) primary antibody at a final dilution of 1:2 000, and with a goat pAb to mouse IgG (Abcam) second antibody at a final dilution of 1:20 000.

Chemicals used include SB 204990 (Thermo Fisher Scientific, Paisley, UK), TOFA, C75, clofibrate, palmitic acid, and SC-236 (Sigma-Aldrich, Dorset, UK), PGD₂, PGI₂, and PGE₂ (Cambridge Bioscience, Cambridge, UK), and SC-51322, TG-4-155, and ER-819762 (Bio-Techne Ltd., Abingdon, UK), and all were reconstituted in dimethyl sulfoxide. TXA₂ (Cambridge Bioscience, Cambridge, UK) was reconstitute in ethanol. PGF_2α (Cambridge Bioscience, Cambridge, UK) and etomoxir and malonyl-CoA (Sigma-Aldrich, Dorset, UK) were reconstituted in E199 medium. Anti-β-actin mouse monoclonal antibody (MAb), anti-FASN goat polyclonal antibody (pAb), anti-COX2 goat pAb, and anti-ACC rabbit MAb (Abcam, Cambridge, UK) were used for Western blot analysis. Donkey pAb to goat IgG, goat pAb to mouse IgG, and goat pAb to rabbit IgG were also purchased from Abcam, Cambridge, UK.