Anti ha hrp antibody

Leaf tissues (60 mg) from Arabidopsis T1 plants or from transiently transformed Nicotiana benthamiana plants were ground in 2x SDS buffer and boiled for 5 min at 95°C. After centrifugation for 10 min at 14000 rpm at 4°C supernatants were digested with Endoglycosidase H (New England BioLabs) as per manufacturer’s suggestion and separated on 8% SDS-PAGE gel. Proteins were detected using anti-HA-HRP antibody (Sigma-Aldrich).

Antibodies against USP11, USP15 and SMAD1 were raised in sheep using GST-tagged proteins as antigens and affinity purified. Anti-SMAD1 antibody for use with Xenopus extracts was from Santa Cruz Biotechnology. Antibody against ALK3 was raised in sheep using His-ALK3(aa200-end) as an antigen and affinity purified. Anti-HA-HRP antibody and anti-α tubulin were from Sigma. Antibodies against phospho-SMAD1/5 (Ser463/465) and 8 (Ser426/428), GAPDH, β-actin and Lamin A/C were from Cell Signaling Technology. Anti-ubiquitin antibody was from Dako. Goat anti-rabbit, mouse and sheep HRP conjugated antibodies were from Pierce. Goat anti-mouse and anti-rabbit IRDye 680LT and 800CW coupled antibodies were from Li-Cor.