Anti gfp ab290

The antibodies used for the immunoblot analysis are as follows: anti-pERK(1/2) (4377T), anti-ERK(1/2) (9102S), and anti–glyceraldehyde phosphate dehydrogenase (2118S) from Cell Signaling Technology. 4G10 was from Millipore (05-321), and anti-GFP (ab290) was from Abcam. Briefly, the cells were stimulated as indicated, and then the cells were lysed using radioimmunoprecipitation assay buffer (9806S) on ice for 10 min and then centrifuged at 4°C and 13,000 rpm. The protein concentration was measured using a Bio-Rad protein assay dye reagent (Bio-Rad, #5000006) following the protocol of the manufacturer. The same amount of boiled proteins was loaded into precast 4 to 20% gradient SDS–polyacrylamide gel electrophoresis (PAGE) (cat#4561096) or 10% precast SDS-PAGE gel for ERK phosphorylation measurement. The gel was run at 110v for 60 to 90 min and then transferred to a nitrocellulose filter membrane at 110v for 60 min. Dry milk (5%) was used to block the membrane at room temperature for 30 min. Then, the membrane was incubated with primary antibody at 4°C overnight, followed by incubation with the secondary antibody at room temperature for 1 hour. Last, the membrane was visualized with standard chemiluminescence.

As described previously, transgenic flies were expanded in vials and bottles containing standard Drosophila media at ~22°C. Larvae, white pre-pupae(WPP) and adults were collected from these expansion bottles. Pupae were obtained by collecting WPP and incubating for 1-2 days at ~22°C. For embryonic collections, 0-8 day old adult flies were transferred to embryo cages capped with apple juice plates. After a 24 hour preincubation, apple juice plates were replaced at appropriate intervals, restricting the collected embryos to their desired developmental stages. For late embryonic stages, collection plates were removed and maintained at room temperature prior to chromatin collection.
Chromatin was collected and immunoprecipitated (IP) using the goat anti-GFP described previously (Zhong et al. 2010 (link); Niu et al. 2011 (link); Kasper et al. 2014 (link); Kudron et al. 2018 (link)). However, due to its limited supply, later experiments used anti GFP Ab290 (Abcam) (Suppl. File 1). In total, ChIP was successfully performed for 677 (604 different TFs) experiments with TFs and another 63 experiments (56 different genes) for nonTFs. Of the 12 tagged TF strains without ChIP, six failed to yield data and for another six, ChIP was not attempted.