To detect the level of autophagy and endoplasmic reticulum autophagy, the GFP–MoAtg8 fusion protein or the GFP–MoSec62 fusion protein were transferred to the wild-type strain and ΔMocbp7 via the ATMT method. The strains were cultured in liquid CM medium for 40 h (25 °C, 150 rpm) then shifted to CM containing 5 mM DTT or tacrolimus (FK506) or were transferred to nitrogen-starved (SD-N) liquid medium. For the protein extraction, the harvested mycelia were collected and shattered in 500 uL of protein lysis buffer (500 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) with 5 uL protease inhibitor (FDbio, Hangzhou, China). GFP-tagged fusion proteins and free GFP were probed with an anti-GFP antibody (HUABIO, Hangzhou, China). Densitometric analysis was performed on ImageJ (x64) 1.8.0 to quantify the degradation level of fusion proteins; GAPDH was used as a control.
Anti gfp antibody
Manufactured by Huabio
Sourced in China
The Anti-GFP antibody is a laboratory reagent designed for the detection and analysis of green fluorescent protein (GFP) in various biological samples. It is a highly specific antibody that binds to GFP, allowing for the visualization and quantification of GFP-tagged proteins or cells.
Automatically generated - may contain errors
3 protocols using anti gfp antibody
1
Analyzing Autophagy in Fungi
2
Investigating phosphorylation-dependent RNS1 localization
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To assay the impact of phosphorylation of RNS1 by Fus3 on its cellular localization, GFP-tagged proteins RNS1-DBD::GFP, RNS1T215A-DBD::GFP, and RNS1S226A-DBD::GFP were constructed. To do this, the coding sequences of RNS1-DBD, RNS1T215A-DBD, and RNS1S226A-DBD were individually inserted into the BamHI/EcoRI sites of the plasmid pPK2-Ptef-GFP-N (26 (link)), and the resulting plasmids were transformed into the WT strain to produce WT-RNS1-DBD-GFP, WT-RNS1T215A-DBD-GFP, and WT-RNS1S226A-DBD-GFP strains and into the ΔFus3 mutant to form ΔFus3-RNS1-DBD-GFP, ΔFus3-RNS1T215A-DBD-GFP, and ΔFus3-RNS1S226A-DBD-GFP strains. The expression and integrity of the fusion proteins were analyzed with Western blot analysis using anti-GFP antibody (Huabio, Hangzhou, China). The subcellular localization of a fusion protein was determined by following the GFP signal. The nuclear was visualized by DAPI (4′,6-diamidino-2-phenylindole) staining.
3
Co-immunoprecipitation of AvrPi9 and OsRGLG5
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The coIP assay was performed as described previously with some modifications (Wang et al., 2020 (link); Shi et al., 2021 (link)). The full-length CDS of AvrPi9 (without the signal peptide) was cloned into the pYBA1132 vector digested by EcoRI and HindIII, and the full-length OsRGLG5 was cloned into the pHY35S-Flag vector digested by KpnI and SalI. The primers used are listed in Supplemental Table 1 . OsRGLG5-Flag was transiently co-expressed with AvrPi9-GFP or empty GFP in N. benthamiana leaves through Agrobacterium-mediated infiltration (Wang et al., 2020 (link)). Total proteins were extracted with protein extraction buffer (150 mM NaCl, 25 mM Tris–HCl [pH 7.4], 1 mM EDTA, 5% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1× Roche protease inhibitor cocktail). After centrifugation at 12 000 g for 10 min, the supernatant was precipitated with GFP-Trap (Chromotek, GTMA-20) according to the manufacturer’s instructions. The immunoprecipitated proteins and input control were detected by western blotting with primary antibodies, including anti-GFP antibody (HUABIO) and anti-Flag (Sigma, A8592), and secondary antibodies (Beyotime, A0208). Individual bands were detected with the ChemDoc Touch Imaging System (Bio-Rad).
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