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8 protocols using control agarose resin

1

PfSET2 Binding to Rpb1 CTD

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Lysates from bacterial cultures expressing HA-tagged PfSET2 fragments and the ySRI were incubated with purified, recombinant Rpb1 CTD in both phosphorylated and unphosphorylated forms in 3% BSA at 4°C for 16 h. Reactions were precleared on control agarose resin (Thermoscientific) and loaded onto Protein A agarose beads (Millipore) for two hours. Bead were washed extensively three times with 50 mM Tris, pH 8.0, 0.5 M NaCl, 0.5% Tween-20 and 10% glycerol. Polyclonal anti-HA antibody (Rabbit; Sigma) was used to immunoprecipitate PfSET2 and any interacting proteins. Immunoprecipitated proteins were separated by SDS-PAGE and the CTD of Rpb1 was detected by Western blotting using anti-Flag antibody.
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2

Disulfide-Preserved NF-κB p50 Profiling

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Nuclear protein was collected using a commercially available Nuclear Extract kit (Active Motif, Carlsbad, CA). Co-IP was performed on the pre-cleared nuclear extract with a goat polyclonal anti-NF-κB p50 antibody (10 μg/column, C-19, Santa Cruz Biotechnology, Dallas, TX) using a commercially available kit (ThermoFisher Scientific, Rockland, IL) according to manufacturer’s instructions. The elution fractions from the IP were then resolved in non-reducing conditions (no DTT added to the sample, preserving disulfide bridges), on a 4–12% Bis Tris gel (Invitrogen, Carlsbad, CA, USA). Western blot analysis and staining with a goat polyclonal anti-NF-κB p50 antibody (1:500, C-19, Santa Cruz Biotechnology, Dallas, TX) was used to identify the disulfide bond preserved protein binding profile of nuclear NF-κB p50 in microglia. Non-eluted nuclear protein was probed with rabbit polyclonal anti-lamin b2 antibody (1:1000 Abcam, Cambridge, MA) on a separate gel as a loading control. Nuclear protein loaded to control agarose resin (ThermoFischer Scientific, Rockland, IL) was used as a negative control.
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3

Mitochondrial Protein Interactions

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For the interaction between MITOSUR-Flag or MITOSURK513A-Flag and Mitok-V5, HeLa cells were transfected with the indicated plasmids. After 48 hours, cells were lysed in CoIP buffer (150mM NaCl, 1% Digitonin, 50mM Tris-Cl pH7.4, 1mM EGTA-Tris pH 7.4 and Complete EDTA-free protease inhibitor mixture). Lysates were centrifuged at 15000xg for 10 min, and supernatant was transferred into new tubes. One mg of proteins was precleared using a control agarose resin (Thermo Fisher Scientific) for 30 min at 4°C. Precleared proteins were incubated with monoclonal α-Flag-agarose-conjugated antibody (Sigma) for 3 hours at 4°C. After 3 washes (10’) in CoIP buffer, 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. The precleared lysate (Input) and the immunoprecipitated (CoIP) fractions were separated and blotted as described above.
For the interaction between endogenous MITOK and MITOSUR, isolated mitochondria from mouse liver or HeLa cells were lysed in CoIP buffer and processed as indicated above. One mg of precleared proteins were incubated with 5 μg of anti-MITOKN-term (Sigma HPA010980) antibody for 3 hours. Protein A-Sepharose beads (GE Healthcare) were added for 1 hour and washed 3 times with CoIP buffer. 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. Results are representative of at least three independent transfections.
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4

Optimized Co-Immunoprecipitation for Viral Complexes

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For both cell lysis and immunoprecipitation buffer, IP buffer was used (50 mM HEPES (pH 7.4), 150 mM NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) NP-40, 1 mM CaCl2), which produced superior results compared to tested HC buffer [50 mM KOAc (pH 6.8), 2 mM MgCl2, 10% (vol/vol) glycerol, 1% (vol/vol) NP-40] in a prior test. Epitope-tagged complexes were pulled down via co-immunoprecipitation. Cell lysates were pre-cleared using control agarose resin (Thermo Scientific) for 1 hour. Subsequently, the lysate was incubated with an anti-HA-Agarose resin (#A2095, Sigma Aldrich) overnight at 4°C constantly rotating. For co-IPs of transfected hepatoma cells, lysates from a whole 10-cm cell culture dish were used. Antigens were eluted by heating up the resin with 4 M guanidine hydrochloride solution pH 8.5 (Sigma Aldrich) for 30 minutes at 900 rpm shaking. Co-immunoprecipitations of E2, NS4B, and ApoE were incubated at 70°C, whereas those for p7 were incubated at 37°C. The samples were stored at −80°C until mass spectrometric analysis. Since guanidine hydrochloride interfered with our control immunoblot analyses, we used 1-µL GlycoBlue coprecipitant (#AM9515, Invitrogen), 5 volumes of ethanol (99% analytical grade), and 0.04 volumes of 2.5 M sodium acetate (pH 5.0) for incubation at 4°C overnight for immunoblot samples.
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5

Capturing EGFR-GFP Complexes via GFP-TRAP

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NHKs stably expressing EGFR-GFP were washed with cold PBS before cold GFP-TRAP lysis buffer (50 mM Tris-HCL pH 7.4, 200 mM NaCl, 2 mM MgCl2, 1% (v/v) NP40, 10% (v/v) glycerol, protease inhibitor cocktail set 1, NaF, phosphatase inhibitor) was added to the cells. Cells were scraped and centrifuged to remove cell debris. 1 : 1 of GFP-TRAP_A beads (Chromotek, Munich, Germany) and control agarose resin (Thermo Fisher Scientific, Waltham, MA, USA) were washed thrice with GFP lysis buffer were washed with GFP lysis buffer before cell supernatants were added to the beads and incubated at 4°C for 2 h. Some of the supernatants were also set aside as input. Afterwards, the beads were washed with lysis buffer without detergent before sample buffer-containing dithiothreitol was added to the beads, boiled and analysed by western blotting.
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6

Mitochondrial Protein Interactions

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For the interaction between MITOSUR-Flag or MITOSURK513A-Flag and Mitok-V5, HeLa cells were transfected with the indicated plasmids. After 48 hours, cells were lysed in CoIP buffer (150mM NaCl, 1% Digitonin, 50mM Tris-Cl pH7.4, 1mM EGTA-Tris pH 7.4 and Complete EDTA-free protease inhibitor mixture). Lysates were centrifuged at 15000xg for 10 min, and supernatant was transferred into new tubes. One mg of proteins was precleared using a control agarose resin (Thermo Fisher Scientific) for 30 min at 4°C. Precleared proteins were incubated with monoclonal α-Flag-agarose-conjugated antibody (Sigma) for 3 hours at 4°C. After 3 washes (10’) in CoIP buffer, 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. The precleared lysate (Input) and the immunoprecipitated (CoIP) fractions were separated and blotted as described above.
For the interaction between endogenous MITOK and MITOSUR, isolated mitochondria from mouse liver or HeLa cells were lysed in CoIP buffer and processed as indicated above. One mg of precleared proteins were incubated with 5 μg of anti-MITOKN-term (Sigma HPA010980) antibody for 3 hours. Protein A-Sepharose beads (GE Healthcare) were added for 1 hour and washed 3 times with CoIP buffer. 50 μl of Laemmli buffer 2X was added to the resin and heated for 5 min at 95°C. Results are representative of at least three independent transfections.
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7

Immunoprecipitation of PBX Protein

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Cells were treated with 60 μmol/L HXR9 or CXR9 for 2 hours and proteins were extracted by using cold immunoprecipitation (IP) lysis buffer (Thermo Fisher Scientific) with protease inhibitor cocktail and pre‐cleared by Control Agarose Resin (26150; Thermo Fisher Scientific). The PBX antibody (sc‐28313 at 20 μg/mL; Santa Cruz Biotechnology) was immobilized using AminoLink Plus Coupling Resin (Thermo Fisher Scientific). Then the pre‐cleared lysate was incubated in antibody immobilized resin with gentle mixing overnight at 4°C. After incubation, the resin was eluted using elution buffer (21004; Thermo Fisher Scientific). Samples of elution buffer were prepared for western blotting analysis as described above.
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8

Immunoprecipitation of HOXB7 Protein

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Proteins were extracted by using cold IP lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease inhibitor cocktail and precleared by the Control Agarose Resin (Thermo Fisher Scientific, 26 150). The HOXB7 antibody (Santa Cruz, sc‐81 292 at 2 μg/mL) was immobilized using AminoLink Plus Coupling Resin. The precleared lysate was then incubated in antibody immobilized resin with gentle mixing overnight at 4°C. After incubation, the resin was eluted using elution buffer (Thermo Fisher Scientific, 21 004). The samples of elution buffer were prepared for western blotting analysis as described above.
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