Streptavidin agarose beads

1×10⁸ HH514-16 cells were induced with 3mM NaB with or without phosphonoacetic acid (PAA) (200 μg/ml) for 36 hours prior to performing iPOND as described previously [29 (link)]. Briefly, cells were pulsed with 10 μM EdU for 15 min, spun down, cross-linked with 1% formaldehyde for 20 min, and quenched with 0.125 M glycine for 5 min. For click chemistry, cells were incubated with 10 μM biotin-azide in click reaction buffer for 2 hours. Nuclei were isolated and digested with 1 μL of micrococcal nuclease (10011, Cell Signaling) at 37°C for 20 min and suspended in cold lysis buffer (1% SDS, 50 mM Tris, pH 8.0), and subjected to sonication using a microtip sonicator to break nuclear membranes. After removing debris, the supernatant was incubated with 100 μL of streptavidin agarose beads (69203, EMD Millipore) overnight at 4°C and washed three times with lysis buffer and one time with 1M NaCl. Protein-DNA complexes were eluted with 2X Laemmli buffer at 95°C for 25 min and subjected to immunoblotting.

circRNA pulldown was performed using Biotin-labeled circPARD3 probes (Sangon Biotech, Shanghai, China). Briefly, FD-LSC-1 cells were lysed by IP lysis buffer supplemented with 1 × protease inhibitor and 1 U/μL RNase inhibitor (ThermoFisher Scientific, Waltham, MA) on ice for 30 min with occasional mixing. The lysate was sonicated for 2 min with 10 s on/off cycles. Then the lysate was precleared with 30 μL streptavidin agarose beads (Cat # S1638, Merck KGaA, Darmstadt, Germany) for 1 h at 4 °C with rotation. Biotin-labeled circPARD3 probes or the negative control (NC) probes were added to the precleared lysate, and the mixtures were incubated at 4 °C overnight. Next, the mixture was incubated with 30 μL streptavidin agarose beads at 37 °C for 1 h. Wash beads-probe-protein complex with 1 mL IP lysis buffer for six times, and then total RNA bound to the beads was extracted, followed by reverse transcription and qPCR detection of circPARD3 and miRNAs.

Cells were incubated for 1 h with 50 µM biotin (Sigma) and 10 ng/ml of HGF (Peprotech). Next, cells were collected and lysed using a RIPA lysis buffer (containing 1% NP-40) supplemented with protease and phosphatase inhibitors (Thermo). To remove the excess of intracellular free-biotin and avoid the subsequent saturation of the streptavidin beads, lysates were filtered by centrifugation using a 3 K Amicon Ultra filter column (0.5 ml, Merck). Filtered lysates were incubated with streptavidin–agarose beads (Merck) for 2 h at 4 °C on a rotating wheel. After the incubation, the beads were washed using 2× RIPA, 1× KCl 1 M, 1× 0.1 M Na₂CO₃ and 1× 2 M urea (Tris-HCl pH 8.0). After the last wash, the beads were covered with a layer of storage solution (2 M urea in 100 mM NH₄HCO₃) and frozen until processed for MS. For WB analysis, purified biotin-labelled proteins were eluted from the beads with electrophoresis sample buffer containing DTT and separated by gel electrophoresis before membrane transfer.

iPOND was performed as described previously^{20 (link),38 (link),63} . Briefly, six 15 cm dishes of HCT116 cells were pulse-labeled for 10 min with 10 μM EdU and then either collected directly or washed and treated with 10 μM thymidine for 1 h or 2 mM HU for 3 h. Cells were then cross-linked in 1% formaldehyde in PBS for 20 min at RT, and cross-linking reactions were quenched using 1 mL of 1.25 M glycine. Following permeabilization in 0.25% Triton X-100 for 30 min at RT, cells were washed and resuspended in click reaction buffer (2 mM CuSO₄, 10 mM sodium ascorbate, and 10 μM biotin azide (Invitrogen) in PBS) for 2 h. Cell pellets were washed, lysed in SDS lysis buffer (50 mM Tris (pH 8.0), 1% SDS with protease inhibitor tablet (Roche)), and sonicated for 20 s at 40% amplitude for a total of 5 pulses. Insoluble material was removed by centrifugation, and lysates were transferred to a new tube and diluted 1:1 with PBS. Lysates were incubated for 3 h at 4 °C with 25 μl of streptavidin agarose beads (EMD Millipore). Beads were washed twice with cold lysis buffer, once with 1 M NaCl, and twice more with cold lysis buffer, prior to elution in 2X SDS loading buffer (5% SDS, 25% glycerol, 150 mM Tris (pH 6.8), 200 mM DTT) for 25 min at 95 °C. Subsequent SDS-PAGE and immunoblotting were performed as previously described.

Whole-cell extracts from a stable HEK293T cell line harboring HTBH-tagged β4 subunits were prepared as in Han et al. (2014) and incubated with HA-tagged PR₂₀ or GR₂₀ peptides (2 µM) 2 h at 4^oC. The resulting proteasome-dipeptide complexes were pull-downed with streptavidin agarose beads (Millipore) for 3 h at 4^oC. Unbound proteasomes in the supernatants were discarded, and the pellets were mixed with 2× SDS sample buffer. Different amounts of samples were resolved by SDS-PAGE/immunoblotting against HA and a proteasome core particle α3 subunit.