Myone streptavidin t1 beads

Myone Streptavidin T1 beads (150 μl; Life technologies) were washed once with 400 μl 1 × TWB (5 mM Tris-HCl (pH 7.5), 0.05 mM EDTA, 1 M NaCl, 0.05% Tween 20), separated on a magnet, and resuspended with 300 μl 2× binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl). Then DNA dissolved in 300 μl 10 mM Tris-HCl (pH 7.4) was added into the bead solution and incubated at RT for 15 min with rotation. The beads were then separated on a magnet and biotinylated DNA was bound to the streptavidin beads.

In situ Hi-C was performed as previously reported 9 (link), 32 (link). Briefly, cells were fixed, lysed, and digested with Dpn II restriction enzyme. Biotin was incorporated into the sticky ends of fragments before ligation. Proximity ligation was carried out with T4 DNA ligase. Then, DNA purification was carried out and the DNA was sheared into fragments. End repair, adenylation, and adapter ligation were performed using NEBNext End Repair Kit (NEB, Ipswich, MA, USA). Biotin-labeled fragments were pulled down using MyOne Streptavidin T1 beads (Life Technologies, Waltham, MA, USA). Hi-C DNA was amplified using the KAPA HiFi Library Amplification Kit (KAPA Biosystems, Wilmington, MA, USA). DNA size selection was performed using AMPure XP beads (Beckman Coulter, Brea, MA, USA). The concentration of the Hi-C libraries was determined using the 2100 Bioanalyzer System.

Cells were lyzed in 50 mM HEPES, 10 mM MgCl₂, 0.1% Triton X-100, 150 mM NaCl with protease inhibitor cocktail. After pre-cleared with MyOne™ Streptavidin T1 beads (Thermo, # 65601) for 1 hr, cell lysates were then co-incubated with biotinylated-ConA (0.15 mM) (Vector, # B1005) and MyOne™ Streptavidin T1 beads for 2 h at 4 °C with rotation. The beads were then washed three times with ice-cold lysis buffer and captured proteins were eluted by Laemmli buffer for western blot analysis. The beads pre-blocked with biotin (0.15 mM) (Sigma, # B4501) served as the control.

To examine the effects of ConA on intracellular and cell surface signaling, cells at ~90% confluency were treated with designated concentrations of ConA (in DMEM, MEM, Ham’s F-12K for Hela, Caco-2, and A549 cells respectively) for 4 h after the full medium was removed. Subsequently, cells were replenished with full medium plus ConA for 15 min for cell signaling activations. The activities of intracellular signaling molecules were examined by western blot using the correlated antibodies. For analysis of cell surface receptor tyrosine kinase activities, cells were lyzed with 50 mM Tris, 150 mM NaCl, 1% SDS with protease and phosphatase inhibitors for 20 min and diluted with equal volume of RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, 0.1% SDS, 1 mM EDTA, pH 7.4, with protease and phosphatase inhibitor cocktail (Roche)). For phosphotyrosine immunoprecipitation, Hela cell lysates were pre-cleared with MyOne™ Streptavidin T1 beads (Thermo, # 65601) at 4 °C for 2 h and then immunoprecipitated with a biotinylated phospho-tyrosine antibody (PY20) (Gibco, # MA12439) at 4 °C overnight. The immunoprecipitated proteins was captured with 30 μl magnetic beads for 1 h at 4 °C and washed three times with ice-cold RIPA buffer. The captured proteins were then eluted from beads with Laemmli buffer and heated at 95 °C for 5 min for western blot analysis.

Nuclear extracts were diluted to 1 mg/mL in Protein Binding Buffer (PBB, 150 mM NaCl, 50 mM Tris HCl pH 8.0, 0.1% NP-40) and incubated with UNC4151 biotinylated-analog for 2 hours at 4°C followed by capture on pre-washed MyOne T1 streptavidin beads (Invitrogen) for 2 hours at 4°C. Supernatant was removed, and beads were washed three times at 4°C in PBB followed by elution of bound proteins. For RNase digestion experiments, extracts pre-incubated with biotinylated compound were divided into two tubes, 100 μg/mL RNase A (Sigma) was added to one tube, and proteins from each tube were captured on T1 streptavidin beads. For western blotting, proteins were eluted in 1X loading buffer with 2-mercaptoethanol at 95°C for 5 min. For proteomics analyses, protein-bound beads were digested with trypsin. Digestions were quenched with 0.1% formic acid and peptides were pressure loaded onto a microcapillary column paced with strong cation and reverse phase resin. A 10-step MudPIT run was performed on an LTQ Velos Pro with in-line Proxeon Easy nLC. The 10 most intense ions identified in MS1 by the mass spectrometer in data dependent acquisition mode were selected for MS/MS fragmentation using collision induced dissociation. Dynamic exclusion was set to 90 sections with a repeat count of 1. Raw data files were matched to the protein database using SEQUEST (Proteome Discoverer 1.4, Thermo).