Anti gfp

The total protein was extracted from N. benthamiana leaves and homogenized in a lysis buffer containing 2% β-mercaptoethanol, 6% sodium dodecyl sulfate (SDS), and 100 mM Tris-HCl (pH of 8.8). The protein samples were individually mixed with SDS loading buffer and boiled for approximately 8 min. Protein samples were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. The blots were incubated in a blocking buffer (5% skim milk in 1×PBS) for 1 h, followed by detection using specific anti-GFP (TransGen Biotech, Beijing, China) or anti-CWMV CP primary antibody and then an HRP-conjugated anti-mouse or anti-rabbit secondary antibody (Abbkine Scientific, California, USA). The detection signal was visualized using an Amersham Imager 680 machine (GE Healthcare BioSciences, Pittsburgh, PA, USA). In addition, the specific primary antibody for detecting CWMV CP was prepared and preserved in our laboratory.

FITC (fluorescein isothiocyanate) and TRITC (tetramethylrho damine) filters were used to assess green fluorescence for GFP and red fluorescence for chlorophyll autofluorescence from chloroplasts. The fluorescence intensity of merged images with orange color revealed the expression of GFP. We selected young leaves of single copy positive plants at 30 days after germination (DAG) to analyze the expression of GFP in transgenic rice with TaAFP-Ba-GFP and TaAFP-Bb-GFP constructs mounted on covered glass slides by an Olympus BX-60 of Confocal Laser Scanning Microscope. We used excitation lasers at 488 and 568 nm to excitate GFP and chlorophyll, respectively; images were collected by FITC and TRITC filters, and single-channel images were superimposed to observe the expression of GFP in leaves. At the same time, Anti-GFP (TransGen Biotech) was selected as a probe to detect the TaAFPB fusion protein in transgenic rice.

The ZmWEE1, ZmPP2Ac‐2, and ZmTE1 genes were cloned into the pCAMBIA1390‐7Myc‐6HIS or pEareyGate 101‐YFP vectors to generate the 35S::ZmTE1‐Myc, 35S::ZmTE1‐YFP, 35S::ZmPP2Ac‐2‐Myc, and 35S::ZmWEE1‐YFP plasmid, respectively. Constructs were then transformed into Arabidopsis mesophyll cells for transient protein expression. Co‐immunoprecipitation (Co‐IP) was performed according to a previous study (Lv et al., 2020 (link)). In brief, Arabidopsis mesophyll cells were harvested and lysed in cell lysis buffer (0.5 mm EDTA; 10 mm Tris‐HCl; pH 7.5; 0.5% NP‐40; 1 mm PMSF; 150 mm NaCl) on ice for 30 minutes with pipetting every 10 minutes. Cell lysates were centrifuged, and the supernatant was incubated with MYC‐Trap magnetic agarose beads (Chromotek, catalog number ytma20, Germany) at 4 °C for 2 h. The beads were washed three times with dilution buffer (10 mm Tris‐HCl; pH 7.5; 150 mm NaCl; 0.5 mm EDTA) and then re‐suspended in SDS loading buffer. The re‐suspended beads were boiled for 10 minutes followed by western blotting using anti‐MYC (Abclonal, catalog number AE010, Wuhan, China) or anti‐GFP (TransGen Biotech, catalog number HT801‐02, Beijing, China) antibody.

For extraction of total proteins, 4- to 5-week-old rosette leaves in soil were ground to a fine powder in liquid nitrogen, and homogenized in two volumes of lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.2% Nonidet P-40, 2 mM DTT, 10% glycerol, protease inhibitor) and centrifuged for 15 min at 12,000 rpm. The supernatant was mixed with 5 × SDS loading buffer and boiled at 95 °C for 5 min. After centrifugation at 12,000 rpm for 1 min, the supernatant containing total proteins was then separated on SDS-PAGE gels. Western blot analyses were carried out using anti-GFP (TransGen Biotech HT801, Beijing, China), anti-MYC (TransGen Biotech HT101), anti-Actin (CWBIO CW0264M), and anti-MPK3 (ABclonal A0228). Western blots were developed with ECL+ (Vazyme E412-01), detected with ChemiDoc XRS+, and quantified using the ImageJ software.

80 μg of above proteins were separated in a 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore, United States). The membrane was rinsed in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) and blocked with 5% fat free milk in TBST at room temperature for 1 h. The membrane was then incubated with anti-GFP (1∶1000 dilution;Transgene, Beijing, China), anti-β-actin, anti-Histone antibody (1∶1000 dilution; Sangon Biotech, China) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1∶1000 dilution; Transgene, Beijing, China). The immunoblots were detected by enhanced chemiluminescence reaction (Engreen Biosystem, China) and measured with densitometry.

Cells were harvested and lysed in cell lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and 1 mM PMSF) on ice for 30 min with pipetting every 10 min. Cell lysate was centrifuged and the supernatant was incubated with MYC-Trap magnetic agarose beads (Chromotek, catalog number ytma-20, Germany) at 4°C for 2 h. The beads were washed three times with dilution buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mM EDTA) and resuspended in SDS loading buffer. The resuspended beads were boiled for 10 min at 99°C and western blotting was followed using anti-MYC (Abclonal, catalog number AE010, MA, United States) or anti-GFP (TransGen Biotech, catalog number HT801-02, Beijing, China) antibody.