Gfp antibody

For co-immunoprecipitation assays, the full length of CsGL2-LIKE-GFP and CsJAZ1-3*HA was inserted between the EcoRI and BamHI restriction sites of pCMBIA1300 (CAMBIA) to create 35S::CsGL2-LIKE-GFP and 35S::CsJAZ1-3*HA. These two vectors were subsequently co-transformed into N. benthamiana leaves as described before (Schütze et al., 2009). CsGL2-LIKE-GFP/pCMBIA1300-HA and CsJAZ1-HA/pCMBIA1300-GFP were used as negative controls. Leaf samples were collected after the plants were cultivated for 72 h, and total proteins were extracted by using lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.5% Nonidet P-40). To inhibit protein degradation, a protease inhibitor cocktail consisting of 20 μM proteasome and inhibitor MG132 was used. The protein mixture was incubated with GFP antibody (Abclonal, China) and Protein G Sepharose (GE Healthcare, UK). Additionally, hemagglutinin (HA) antibody (Abclonal, China) was used in western blots for protein detection. Gene-specific primers are listed in Supplementary Table S1.

Cell lysates were prepared and separated by sodium dodecyl sulfate (SDS) gel electrophoresis, electrotransferred to a nitrocellulose membrane (GE), blocked with 5% non-fat milk in 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20 (v/v) (TBS-T buffer) for 60 min at room temperature, and incubated with the primary antibodies. The primary antibodies used were anti-LAMP1 (Proteintech, China; 21997-1-AP), TfRC antibody (Proteintech; 10084-2-AP) was used at a 1:1,000 dilution, the GFP antibody (Abclonal, China; AE012) was used at a 1:1,000 dilution, Flag M2 antibody (Sigma, F3165) was used at a 1:3,000 dilution, FTH antibody (Abcam, ab75973) was used at a 1:1,000 dilution, glutathione peroxidase 4 (GPX4) antibody (Abcam, ab125066) was used at a 1:1,000 dilution, actin antibody (Absin, China; abs125702) were used at a 1:1,000 dilution, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Proteintech; 60004-1-AP) was used at a dilution of 1:10,000. Secondary antibodies used were anti-mouse IgG conjugated with peroxidase (POD) (Proteintech; SA00001-1) at a 1:10,000 dilution or anti-mouse IgG, 800 (LI-COR Biosciences) at a 1:250,000 dilution, anti-rabbit IgG conjugated with POD (Proteintech; SA00001-2) at a 1:10,000 dilution or anti-mouse IgG, 800 (LI-COR Biosciences). Relative protein amounts were determined using ImageJ.