Rabbit anti ha antibody

The synthetic oligonucleotides (5′-GCC CAT AGC CAT CAG TCA CAA CTA CCA-3′) containing one TLX-binding site (5′-CAG TCA-3′) were annealed and labeled with ³²P-dCTP as a probe. Gel-shift assays were performed essentially as previously described (Zhang et al., 2006 (link); Qin et al., 2013 (link)). Briefly, in vitro translated TLX protein was incubated with ³²P-labeled probes (5 × 10⁵ c.p.m.) in binding buffer for 20 min at RT. Antibody-dependent supershift assays were performed by adding 1 μg rabbit anti-HA antibody (Santa Cruz) followed by incubation for 15 min at RT. An equal amount of normal rabbit serum was included as a control. Non-labeled double strand oligonucleotides were used for competition assays. ChIP assays were performed using NSCs transduced with retrovirus expressing either GFP or HA-tagged TLX. NSCs were fixed with 1% formaldehyde for 10 min at RT and the reaction quenched with 0.25 M glycine for 5 min at RT. ChIP was performed as previously described using a rabbit anti-HA antibody (Santa Cruz) (Qin et al., 2013 (link)). Purified DNAs were amplified with the following primer pairs: 5′-TTG TGA CTG ATG GCT ATG GG-3′ and 5′-AAG GGA TTG TGT CTC GCA TAC-3′ for the TLX-binding site and 5′-GGG TTT CAA AGG ATG GTC AAT G-3′ and 5′-AGG CTG GTC TCA AAC TTC TG-3′ for a distal control site.

Cell lysates from HEK293T cells were collected and lysed in NP40 lysis buffer (500 mM Tris HCl, pH 8, 500 mM MgCl₂, 2.5 M NaCl, 0.25 M EGTA, pH 8, 5% NP40) supplemented with 1 mM Na₃VO₄ and 50 mM NaF and 1 tablet of cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail per 10 mL buffer (Roche). For co-IP using the HA-tag, proteins were incubated with rabbit anti-HA antibody (Santa Cruz Biotechnology, sc-805) for 2 hours, rotating at 4°C followed by overnight incubation with M-280 sheep anti–rabbit IgG Dynabeads (Thermo Fisher Scientific, 11203D). Beads were isolated using a magnetic block and washed 5 times with washing buffer (50 mM Tris HCl, pH 7.4, 250 mM NaCl), rotating at 4°C for 30 minutes before they were collected in 1× NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, NP0007) supplemented with 1:100 DTT and stored at –20°C.

Cells or brains were lysed in sample buffer [50 mM Tris-HCl, 1% w/v SDS, 1% 2-mercaptoethanol, 15% glycerol, 0.01% bromophenyl blue, protease inhibitor (Roche), phosphatase inhibitor (Sigma), pH 6.8]. Then, the lysates were separated on SDS-PAGE and transferred to PVDF membranes. Membranes were incubated in blocking buffer (5% nonfat milk, 0.1% tween 20 in TBS) for 1 h at room temperature and followed by incubating overnight at 4 °C with rabbit anti-HA antibody (Santa Cruz) and rabbit anti-GAPDH antibody (Cell Signaling Technology). After washing 3 times, membranes were incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG (Promega) and detected with Western Lightning Ultra (PerkinElmer).