Histone h3k4me3

Whole cell lysates were prepared by lysing cells with RIPA buffer (0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 25 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor mixture and PhosSTOP (Roche). Histones were prepared by acid extraction as previously described (Choi et al., 2016 (link)). Proteins were quantified with Bradford assay (Bio-Rad). Equal amounts of proteins were separated with SDS-PAGE and transferred to nitrocellulose membranes. To visualize equal protein loading, blots were stained with Ponceau S. Blots were incubated in 5% non-fat milk in TBST, probed with primary antibodies to HA (1:1,000, Cell Signaling Technology, Cat# 2367), CtBP2 (1:1000, BD Biosciences, Cat# 612044, RRID:AB_399431), Mek1/2 (1:1,000, Cell Signaling Technology, Cat# 9122), alpha-tubulin (Sigma, Cat# T5168), MLL1 (1:1,000, Cell Signaling Technology, Cat# 14197), CtBP1 (1:1,000, BD Biosciences, Cat# 612042, RRID:AB_399431), Myc tag (1:1,000, Cell Signaling Technology, Cat# 2276), V5 (1:1,000, Invitrogen, Cat# R950-25), Histone H3K4me3 (1:1,000, Cell Signaling Technology, Cat# 9751, RRID:AB_2616028), and Histone 3 (1:1,000, Cell Signaling Technology, Cat# 5427) and then were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by enhanced chemical luminescence (Cytiva or Pierce).

Flies were reared at 25°C and embryos were fixed and stained according to standard protocols (Crocker et al., 2015 (link)). Primary antibodies were detected using secondary antibodies labeled with Alexa Fluor dyes (1:500, Invitrogen). In situ hybridizations were performed using DIG or biotin-labeled, antisense RNA-probes against a reporter construct RNA (lacZ) or the first intron of svb or fkh. DIG-labeled RNA products were detected with a DIG antibody: Invitrogen, 9H27L19 (1:200 dilution) and biotin-labeled RNA products are detected using a biotin antibody: Pierce, PA1-26792 (1:200).
The following primary antibodies for proteins were used at the indicated concentrations:
Ubx: Developmental Studies Hybridoma Bank, FP3.38-C (1:20)
Hth: Santa Cruz Biotechnology (dN-19), sc-26186 (1:50)
Eve: Developmental Studies Hybridoma Bank, 2B8-C (1:20)
AbdA: Santa Cruz Biotechnology (dN-17), sc-27063 (1:50)
En: Santa Cruz Biotechnology (d-300), sc-28640 (1:50)
RNA PolII RPB1 (Ser5 phosphorylated): BioLegend, (920304), (1:200)
Histone H3K27me3: Active Motif, 39157 (1:200)
Histone H3K4me3: Cell-signaling technology C42D8 (1:200)
LacZ: Promega anti-ß-Gal antibody (1:1000)