Gfp antibody

Overnight cultures of cells with plasmids encoding YfiN and its mutants (I_ps, R260A, GGAAF) were diluted 1:100 in M9M and propagated with 0.02% arabinose for 3 h. Cells were collected and then resuspended in a lysis buffer (12.5 mM Tris pH 6.8, 4% SDS) with a final concentration of 2.5 × 10^9/mL cells. Upon cell lysis using a heat block (100°C), 1 × 10^7 cells were loaded into each lane on a SDS-gel. Proteins were then transferred to a nitrocellulose membrane and blocked with 5% non-fat dry milk (NFDM) in Tris-buffered saline with 20% Tween (TBST) for 1 h. The membrane was incubated overnight with a 1:1000 dilution of either GFP antibody (Sigma) or FLAG antibody (Sigma Monoclonal FLAG antibody M2) in TBST with 5% NFDM (wt/vol), and then washed 3 times with TBST every 10 min intervals. This step was followed immediately by an hour incubation with 1:5,000 dilution of Goat α-mouse-HRP (Bio-Rad) in TBST. ECL Select (Amersham; chemiluminescence) was used for a development and the blot image was taken using ChemiDoc (G:BOX).
ImageJ software was used to measure the difference in pixel intensities to compare the difference in the levels of expression.

ChIP assay was used to study the enrichment of CaWRKKY40 in the promoters of its target genes following the method as previously described [85 (link)]. Briefly, GV3101 cells containing 35S:CaWRKY40-GFP or 35S:GFP were infiltrated into pepper leaves of TRV:CaSTH2 or TRV:00 plants upon RSI or HTS. Chromatin was extracted from leaves infected with Agrobacterium at 48 hpi and randomly broken into fragments of 300–500 bp using sonication. The fragment mixture was incubated with magnetic beads fused with GFP antibody (Sigma-Aldrich, St. Louis, MO, USA) for more than 2 hpi, then the DNA was collected from the magnetic beads and purified. Before confirmation by PCR and qPCR, the DNA fragments were further used in the ChIP-qPCR assay.

Antibodies for Western blotting were as follows: CAPS antibody raised against the full-length protein in rabbit, purified by protein A–agarose chromatography, and used at 1:1,000; GAPDH antibody purchased from Life Technologies (catalogue number AM4300) and used at 1:4,000; Rbcn3α antibody purchased from AbCam (catalogue number ab234771) and used at 1:000; Rbcn3β antibody purchased from Sigma-Aldrich (catalogue number HPA042074) and used at 1:1000; V-ATPase V0A antibody purchased from Sigma-Aldrich (catalogue number HPA022144) and used at 1:1000; V-ATPase V1A antibody purchased from AbCam (catalogue number 137574) and used at 1:1000; GFP antibody purchased from Sigma-Aldrich (catalogue number G1544) and used at 1:2000; and HA antibody purchased from Sigma-Aldrich (catalogue number H3663) and used at 1:2000. Rbcn3β antibody for immunofluorescence was purchased from Protein Tech (catalogue number 24431-1-AP) and used at 1:50, and Rbcn3α antibody for immunofluorescence was obtained from Sigma-Aldrich (HPA039375) and used at 1:50.

Cortex samples from 12-week old 5XFAD; Becn1^FA/FA; GFP-LC3 mice were dissected and homogenized in 1 ml cold lysis buffer pH 7.4 containing 250 mM sucrose, 1 mM EDTA, 10 mM HEPES, halt proteinase inhibitor cocktail (ThermoFisher Scientific), and halt phosphatase inhibitor cocktail (ThermoFisher Scientific), using a Dounce tissue grinder (Wheaton). The lysate was then passed 15 times through 27-gauge needle. GFP-based immunoisolation was performed using Dynabeads Protein G (ThermoFisher Scientific). The lysate was centrifuged at 1,000 x g for 10 min at 4°C. The post-nuclear supernatant fraction was centrifuged at 20,000 x g for 20 min and the supernatant fraction was discarded to remove residual cytosolic GFP-LC3 [61 (link)]. The pellet fraction was resuspended in 250 μl lysis buffer and was incubated for 2 hours at 4°C with 40 μl of Dynabeads, preincubated O/N with GFP-antibody (Sigma, G1544). The beads were then washed 4 times with wash buffer (150 mM NaCl, 250 mM sucrose, 1 mM EDTA, 10 mM HEPES) using the magnetic Separator DynaMag^TM-2 (ThermoFisher Scientific). Immunoprecipitates were eluted with lysis buffer containing 1X sample buffer and analyzed by SDS-PAGE.

Immunoprecipitation experiments in transiently transfected HEK 293 cells were performed as previously described [30 (link),47 (link)]. Briefly, forty-eight hours after transfection, cells were lysed in 50 mM Tris HCl, 150 mM NaCl, and 1% NP40 supplemented with protease inhibitors. Pre-cleared protein extracts were incubated overnight on a rotary wheel at 4 °C with the GFP antibody (Sigma-Aldrich, Munich, Germany, Erembodegem, Belgium) and protein-A/G agarose beads (Santa Cruz Biotechnology, Heidelberg, Germany). Immunoprecipitates were collected by a 5 min spin at 2000 rpm, washed three times in PBS, and analyzed by Western blot analysis using SERCA2 (Cell Signaling Technology, Leiden, The Netherlands) or GFP (Sigma-Aldrich, Munich, Germany) antibodies.

To verify protein expression, a fraction of the cells was harvested and lysed in lysis buffer containing 50 mM Hepes (pH 7.5), 250 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM dithiothreitol (DTT), 1 mM PMSF, and Protease Inhibitor Cocktail (Roche). GFPMBP-BRCA2 protein was detected from GFP-trap (ChromoTek) immunoprecipitates by immunoblotting with GFP antibody (Sigma G1544, 1:500).

ChIP-PCR assay was performed as previously described (Yuan et al., 2019a (link),b (link); Yuan X. et al., 2021 (link)). Briefly, ∼4 g leaves from ONAC096-OE12 plants were harvested and immediately crosslinked in 1% formaldehyde by vacuum infiltration for 30 min. After an addition of 125 mM glycine to stop the cross-linking, the chromatin DNA was extracted and fragmented to 200∼500 bp by sonication (Bioruptor Plus, Diagenode, Belgium). The fragmented DNA was pre-cleaned with Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, Waltham, MA, United States) and an aliquot (10%) of the chromatin DNA was used as an input control. The remaining pre-cleaned chromatin DNA was incubated overnight with GFP antibody (Sigma-Aldrich, Saint Louis, MO, United States) or with pre-immune (Pre) serum (GenScript, Nanjing, China) (a negative control). After reversal of the cross-links, the immunoprecipitated DNA and input DNA were extracted by phenol/chloroform extraction. PCR analysis of ChIP DNA and input DNA were carried out using gene-specific primers (Supplementary Table 1).