Flag tagged beads

Neuro-2a (ATCC CCL-131) cells were cultured in 145 mm tissue culture treated dishes (Greiner, #639960) at 37°C with 5% CO2 and transfected with Lipofectamine 3000 reagent (Thermo, #L3000001) with the following plasmid combinations: Fezf2-his-myc + Tle4-flag-myc + GFP-his-myc, Fezf2-his-myc + GFP-his-myc, Tle4-flag-myc + GFP-his-myc, or GFP-his-myc alone. 24 hours later, cells were harvested, and nuclear extract was isolated using the Active Motif Nuclear Complex Co-IP kit (cat. #54001). The extracts were immunoprecipitated overnight at 4°C with either Flag-tagged beads (Sigma, #M8823) or GFP antibody (Rabbit, Invitrogen A11122) and eluted with 0.1M glycine, pH 2.5 at RT for 30 minutes with occasional agitation. The samples were denatured at 100°C for 5 minutes in 5x sample buffer, run on an 8% SDS-PAGE at 70V for 2 hours, transferred to PVDF (Sigma, IPVH85R) at 150mA for 90 minutes, and blocked for one hour in 1% non-fat dry milk-TBST. 2ug of Fezf2 (Rabbit, IBL F441), Tle4 (Mouse, Santa Cruz Biotechnology sc-365406), GFP (Chicken, Aves Labs GFP-1020), or Myc (Goat, Abcam ab9132) primary antibodies were added and incubated overnight at 4°C on an orbital shaker. The blot was developed with Li-Cor secondary antibodies (#926-32212, 926-32214, 926-68073, 926-68072) or Alexa 488 for one hour and images were processed using ImageStudioLite.

Protease Inhibitor Cocktail, Aurora-A Inhibitor-I (AAI), nocodazole, Phosphatase Inhibitor Cocktail and Flag-tagged beads were purchased from Sigma-Aldrich. 1,4-dithiothreitol (DTT) and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Acros Organics. Insect cell-expressed Aurora-A was purchased from Invitrogen. The cDNA reverse transcription kits and TurboFect reagent were purchased from Thermo Fisher Scientific. Hygromycin B and the Midi plasmid extraction kits were obtained from Roche. PVDF membrane, iCRT3 and ECL were purchased from Merck Millipore. The NTA-beads were purchased from QUIAGEN. The protein G beads for immunoprecipitation were obtained from GE. RQ1 (RNase-free DNase) was purchased from Promega. The SYBR Green reagents (the KAPA SYBR FAST qPCR kit Master Mix (2X) ABI Prism) were obtained from KAPA.

Standard methods of Western blotting and immunoprecipitation were used. Briefly, cells were lysed using RIPA buffer (Beyotime, Shanghai, China) and the supernatant was resolved by SDS-PAGE after centrifugation and transferred to PVDF membranes for Western blotting. Then, the membranes incubated with ECL luminescence reagent (Meilun Biotechnology Co., Ltd, Dalian, China) and exposed using Tanon 5200 Chemiluminescence Imaging System (Tanon Technology Co., Ltd, Shanghai, China).
The glutathione S-transferase (GST) pull-down assays and immunoprecipitation experiments were performed as described before [21 (link)]. The supernatant was incubated FLAG-tagged beads (A2220; Sigma-Aldrich, St. Louis, MO, USA) or HA-tagged beads (11815016001; Roche, Mannheim, Germany) or Glutathione Beads (SA008005; Smart-Lifesciences Biotechnology Co., Ltd, Changzhou, China) for 4 h. The precipitates were then washed five times and analyzed by Western blotting.