Dynabeads protein a magnetic beads

For IP of endogenous GLI1, A549, 1321N1 and 293FT adherent cells were lysed in IP buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP-40, 0.5% TritonX-100, 0.1% SDS, 5 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Lysates were incubated on ice for 30 min and cleared by centrifugation at 14000 rpm for 25 min at 4 ^oC. Endogenous GLI1 was immunoprecipitated using GLI1 V812 Ab (5 μl per reaction) and protein A Sepharose beads (GE Healthcare) or Dynabeads protein A magnetic beads (Invitrogen) at 4 ^oC overnight. The immunocomplexes were washed with TBS and subjected to WB (1/5 of the sample) or MS analysis (4/5 of the sample).
293FT cells were transfected using 2 μg of the constructs encoding Flag-tagged GLI1FL, GLI1ΔN, PKA, and ULK3, incubated for 2 days and lysed in 50 mM Tris HCl, pH 7.4 supplemented with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100 for 30 min on ice. The lysates were incubated with Anti-Flag M2 Affinity Gel (Sigma-Aldrich). Endogenous SUFU was immunoprecipitated using -SUFU Ab (C-15) conjugated to agarose (Santa Cruz Biotechnology). The obtained immunocomplexes were washed with TBS and subjected to WB and in vitro kinase assay.

Cells were rinsed in Dulbecco’s phosphate-buffered saline (DPBS), harvested, and lysed in Pierce IP Lysis Buffer (Thermo Scientific) supplemented with complete protease and PhosSTOP phosphatase inhibitor cocktails (Roche) on ice for 30 min. BCA protein assays were performed to quantitate protein according to instructions supplied by the manufacturer (Pierce). Protein lysates (450 μg) were incubated with 1 μg of rabbit monoclonal antibodies against C8A or C8B at 4°C overnight. Rabbit IgG (1 μg, Jackson ImmunoResearch Laboratories) was used as a negative control. The next day, Dynabeads Protein A magnetic beads (Invitrogen) were added, and the mixtures were gently mixed at 4°C for 4.5 h. Thereafter, the beads were isolated and washed with wash buffer (phosphate buffered saline (pH 7.4) with 0.02% Tween 20 in presence of protease and phosphatase inhibitors cocktails), and the protein level of C8A, C8A variant, C8B, C8B variant and C8G were analyzed by western blot.

Immunopurification of peroxisomes was performed essentially as described in Kikuchi et al. [26 (link)]. The peroxisome-enriched fractions from OptiPrep gradient were incubated with Dynabeads Protein A magnetic beads (Invitrogen) linked with rabbit polyclonal anti-PMP70 antibody (Sigma-Aldrich, P0497) for 4 h at 4 °C. Bead complex was collected by placing the reaction tube in a magnet stand. The complex was washed three times with PBS.

To investigate Top2a interaction partners, co-immunoprecipitation was performed from U2OS Nuclear Extract (15 mg/ml total protein). Top2a was immunoprecipitated using an anti-Top2a antibody (ab12318; Abcam) immobilized on Dynabeads Protein A magnetic beads (c/n 10001D; Thermo Fisher Scientific) according to the manufacturer’s instruction. Antibodies were cross-linked to beads using DPM (c/n 21666; Thermo Fisher Scientific) as recommended by the manufacturer. Beads were blocked with BSA in PBS overnight. 100 μl NE was diluted by dilution buffer (25 mM Tris HCl, pH 7.9, 12.5 mM MgCl₂, 10% glycerol, and 0.03% NP40) to a final KCl concentration of 150 mM and treated by 500 U of benzonase (E1014; Sigma-Aldrich) for 30 min at 4°C. 25 μl of the beads were added, and the suspension was incubated on a rotating wheel for 1 h at 4°C. Beads were washed three times with 100 μl wash buffer (25 mM Tris–HCl, pH 7.9, 150 mM KCl, 12.5 mM MgCl₂, 10% glycerol, and 0.03% NP40) and proteins were eluted by incubation in 1× LDS sample buffer (c/n NP0007; Thermo Fisher Scientific) at 65°C for 10 min. Immunoprecipitated proteins were analyzed by Western blot using anti-UBF, anti-RPA49, and anti-Top2a antibodies (sc-9131; Santa Cruz; 611413 BD Transduction; and ab12318; Abcam).

Fly heads were fixed with 1.8% (v/v) formaldehyde for 30 min at room temperature, homogenized, resuspended in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% w/v Triton X-100, 0.1% w/v SDS, 0.1% w/v DOC). Staged embryo chromatin immunoprecipitation (ChIP) material (cycle 11 to cycle14) was prepared according to (Loubiere et al. 2017 (link)). Crosslinked material was sonicated after preparation in 4 ml of 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0 for 30 min with a Branson 450 digital sonifier (45 cycles of 20 s on–40 s off). The sonicated lysate was clarified by centrifugation, preabsorbed by incubation with Dynabeads™ protein A magnetic beads (Thermo Fisher Scientific) and incubated with 7 μg polyclonal antibodies (α-H3K9ac, α-H3K27ac, α-H4K16ac) overnight at 4 °C. Antibody complexes were bound to protein A-Sepharose magnetic beads. Precipitated DNA was recovered and dissolved in 150 μl water. Control mock immunoprecipitations were done in parallel without antibodies. Real-time PCR analysis was performed according to previous studies (Dellino et al. 2004 (link); Rudolph et al. 2007 (link)) and 5 μl DNA from each sample was amplified in 20 μl reactions with 2x SYBR Green Super Mix (Bio-Rad). All primer sequences used in the studies are listed in (Rudolph et al. 2007 (link)).

Total nucleic acids were extracted from MEF by SDS/Proteinase K treatment at 37 °C followed by phenol-chloroform extraction and ethanol precipitation. Free RNA was removed by RNAse A treatment. DNA was fragmented overnight using HindIII, EcoRI, BsrGI, XbaI, and SspI and pretreated, or not, with 40 U RNase H1 (NEB, 5000 U/ml). For DRIP, R-loops were immunoprecipitated using 6 μg DNA-RNA hybrids antibody (Kerafast, Cat. ENH001) per 10 μg of digested DNA in 500 µl IP buffer. Bound R-loops were recovered by addition of 50 μl pre-blocked dynabeads protein A magnetic beads (Thermo Fisher Scientific) followed by two washes and elution in an EDTA/SDS-containing buffer. DNA fragments were treated with Proteinase K and recovered with a QIAquick PCR purification kit (Qiagen). Validation of the DRIP was performed by qPCR. Primer pairs used for DRIP analysis are described in Supplementary Information, Supplementary Table 1.

Total proteins were extracted from the PMVECs using IP buffer, including 0.5M EDTA PH 8.0 (Sangon Biotech, China), NP-40 lysis buffer (Beyotime, China), 1M Tris-HCL PH 7.5 (Beyotime, China), Nacl (Sigma, Germany), and 500 mm DTT (Beyotime, China) for 2 h at 4^oC. We took 200 μg of the whole-protein lysate as the input and heated it at 100^oC for 10 min to denature the protein. Then, we separated the remaining total protein lysate into four tubes and added 10 μL Dynabeads^® Protein A magnetic beads (10004D, Thermo Scientific, USA) respectively to pre-clearance for 2 h shaking with low speed at 4^oC. Next, Rabbit mAB IgG (Magnetic bead conjugate) (#8726, Cell Signaling Technology, USA), and KLK10 (PAA697Mu01, Clone, Hubei, China), LRG1 (PAB934Mu02, Clone, Hubei, China) antibodies were added to the supernatant and transferred mixture for 16–24 h shaking with low speed at 4^oC. A day later, magnetic beads (20 μL per tube) were added to the upper mixtures of KLK10 or LRG1 and shaken at 4^oC for 2 h. The beads were resuspended with equal proportions of IP and Loading to obtain the IP mixture. Finally, we heated the IP mixture at 100^oC for 10 min to denature the protein and used the magnetic frame to absorb the magnetic beads and leave the supernatant for immunoblotting.