Anti tubulin antibody

For total protein extraction, equal amounts of 7-day-old seedlings were grounded into fine powder as described above, and then resuspended in 2× SDS buffer. The samples were lysed on ice for 30 min, then centrifuged at 18,514 g for 10 min at 4°C. The supernatants were transferred into new tubes, and heated in boiling water for 10 min. 10 μL of total protein extracts were separated via 10% SDS-PAGE and then transferred to Protran nitrocellulose membranes (Whatman). The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in Supplemental Figure S2B, and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in Figure 5, A and H at 4°C overnight. The secondary antibodies used were Goat anti-mouse lgG (H + L) HRP (ShareBio; SB-AB0102; 1:5,000) or Goat anti-Rabbit lgG (H + L) HRP (ShareBio; SB-AB0101; 1:5,000). All antibodies were diluted in 5% skim milk powder (in PBS containing 1% Tween-20). The images were taken using the BIO-RAD ChemiDoc Imaging System. The integrated optical density (IOD) values of VLG and tubulin protein band signals in Figure 5, A and H were quantified using Image Lab software (Bio-Rad) (Ye et al., 2019 (link)).