Active tgfβ1

Samples were prepared as follows: 10 ml 100 ng/ml active TGFβ1 (Bio-Techne) in PBS. 10 ml 50 mg/ml VA extract in PBS. 5 ml 100 ng/ml active TGFβ1 in PBS plus 5 ml 50 mg/ml VA extract in PBS, mixed for 4 h on a rotator at RT. Samples were loaded using a 50 ml Superloop (Cytiva) and run on an ACTApurifier 10 system (Cytiva) using the equipment, reagents and programme described^{29 (link)}. Fractions were assayed for TGFβ1 using an ELISA as described.

Leaf extract (1 mg/ml in PBS), TGFβ1 antibody (5 µg/ml in PBS; clone 1D11 Bio-Techne), parthenium (1 mg/ml in chloroform; Sigma), artemisinin (1 mg/ml in chloroform; Sigma), full length Pisum sativum L. (Leguminosae) chlorophyll a-b binding protein AB96 (at indicated concentrations; mybiosource TTKKVASSSSPWHGPDGVK YLGPFSGESPSYLTGEFPGDY GWDTAGLSADPETFAK NRELEVIHSRWAMLGAL GCVFPELLSRNGVKFG EAVWFKAGSQIFSEGGL DYLGNPSLVHAQSIL AIWATQVILMGAVEGYRI AGGPLGEVVDPLYPG GS FDPLGLAEVPEAFAE LKVKELKNGRLAMF SMF GFFVPAIVTGKGPLEN LADHLADPVNNN AWSYATNFVPGK), soybean trypsin inhibitor (at indicated concentrations; Sigma) or vehicle (PBS or chloroform) were plated on a high binding 96 well flat-bottomed plate (Greiner) and left at RT overnight. The next day the plate was washed × 3 in PBS + 0.05% Tween-20 (Sigma) before being blocked with 5% Tween-20 in PBS. Active TGFβ1 (Bio-Techne), IL-10 (Bio-Techne), IL-12p70 (Bio-Techne) or latent TGFβ1 (Bio-Techne) were added at indicated concentrations before being incubated for 2 h at RT. The supernatant was removed and placed immediately in a relevant ELISA for assaying; this formed the ‘unbound fraction’. The plate was washed × 3 in PBS + 0.05% Tween-20 before being treated with 50 µl 1 M HCl for 30 min at RT to elute any bound protein. This ‘bound fraction’ was neutralised using 50 µl 1.2 M NaOH/0.5 M HEPES and immediately assayed in a relevant ELISA.