Anti gfp 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)).

Western blotting was performed as previously described (Yan et al., 2021a (link)). Total protein was extracted from N. benthamiana leaf tissue samples. An anti‐GFP antibody (Abways) was used as the primary antibody. Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG (Sigma‐Aldrich) was used as the secondary antibody. SuperSignal West Dura Extended Duration Substrate solution (Thermo Fisher Scientific) was used as the HRP substrate. The reaction signal was detected using a chemiluminescent imaging and analysis system (Sage).

For the CIP treatment, VLG-GFP was immunoprecipitated as described above. The GFP-conjugated beads were washed by NB1 and CIP buffers (three times each) and then incubated with 2 μL Quick CIP (New England Biolabs) in 500 μL CIP buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM DTT, 1 mM PMSF, and 0.1% NP-40) for 60 min at 37°C. The beads were washed six times with CIP buffer. For the GSK3β treatment, VLG-GFP was immunoprecipitated as described above. The GFP-conjugated beads were washed six times with NB1 buffer and then incubated with 2 μL GSK3β in 300 μL GSK3β assay buffer for 60 min at 37°C as recommended by the manufacturer (SignalChem). The beads were washed six times with NB1 buffer and mixed with loading buffer, and then boiled at 100°C for 10 min. A 20-μL aliquot of each boiled sample was analyzed by immunoblotting using an anti-GFP antibody (Abways; cat. no. AB0005; 1:3000 dilution).