Protein g magnetic dynabeads

Immunoprecipitation of MUC17-3TR was performed on ice. Magnetic Protein G Dynabeads (Life Technologies) were washed in PBS and coated with anti-MUC17C1 antibody in PBS for 5 h at 4°C. Unbound antibody was removed by washing 5× with 1% (v/v) Igepal CA-630 (Sigma) in PBS. Alternatively, pre-coated anti-c-Myc Agarose Beads (Sigma, A7470) were used. Cells were harvested by scraping and lysed in 50 mM Tris pH 7.5, 1 M NaCl, 10 mM MgCl₂, cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche), 5% (v/v) glycerol, 1% (v/v) Triton-X 100, 1 mM EDTA and 1 : 100 phosphatase inhibitor cocktails 2 and 3 (Sigma). Debris was removed by centrifugation at 20 000×g and 4°C for 10 min and supernatant bound to anti-MUC17C1 coated protein G Dynabeads or anti-c-Myc Agarose Beads at 4°C overnight. Unbound material was removed by washing in 1% (v/v) Igepal CA-630 in PBS.

20 µL reactions containing 2.5 µM rOGT and 2.5 µM rTET1 CD wt or D2018A were assembled in binding buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.02% Tween-20) and pre-incubated at room temperature for 15 min. TET1 antibody (Millipore 09 – 872) was bound to magnetic Protein G Dynabeads (Invitrogen), and beads were added to reactions following pre-incubation. Reactions were bound to beads for 10 min at room temperature. Beads were washed 3 times with 100 µL binding buffer, and bound proteins were recovered by boiling in SDS sample buffer and analyzed by SDS-PAGE and coomassie stain.

Cells were lysed in CHAPS buffer (40 mM HEPES pH 7.4, 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail. Lysates (300 μg) were incubated with antibody (1 μg of anti-mTOR, STAT6, eIF4E or isotype control antibodies) at 4 °C overnight. Magnetic protein G Dynabeads (Invitrogen) were then added, and tubes were rotated for an additional 2 h. After washing five times with lysis buffer, proteins were eluted from beads with 2X Laemmli buffer at 95 °C for 10 min, followed by resolution by SDS-PAGE.

Brain homogenates from a block of tissue containing the entire cerebral contusion were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich), followed by sonication and centrifugation at 15,000 × g for 30 min. Supernatants containing protein complexes were incubated with anti-RIPK3 (Prosci, #2283) or anti-RIPK1 (BD bioscience, #610458) antibodies conjugated to Magnetic Protein G Dynabeads (Thermo Fisher Scientific) at 4 °C overnight. Beads were washed three times with washing buffer and proteins were eluted in 1X loading buffer and processed/ for western blot.

SMARCA4 ChIP-seq was performed essentially as described previously^{57 (link)}. Briefly, cells were first crosslinked in 2 mM disuccinimidyl glutarate (ThermoFisher) in PBS for 30 min and then in 1% formaldehyde in the medium for 10 min at room temperature. The cells were quenched with 0.125 M glycine for 5 min and washed twice in ice-cold PBS. Chromatin was isolated and prepared using a ChIP-qPCR Kit (Chromatrap) and sonicated using a Bioruptor Plus (Diagenode) to an average DNA size of 150–400 base pairs. Magnetic protein G Dynabeads (ThermoFisher) were washed with PBS that contained 1% w/v BSA (Sigma-Aldrich), incubated with 5 µg of ChIP-grade antibody against SMARCA4 (Abcam, ab110641) for 1 h at room temperature and washed five times with PBS that contained 1% w/v BSA. Solubilized chromatin from 5 × 10⁶ cells was immunoprecipitated with antibody conjugated beads in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Igepal CA-630 and 0.5% sodium deoxycholate) for 12 h at 4 °C. Magnetic beads were washed 5× with RIPA buffer and chromatin was eluted. After crosslinking reversal, RNAase A (Ambion) and proteinase K (ThermoFisher) treatment, ChIP DNA was extracted using a Min-Elute purification kit (Qiagen). Sequencing libraries of ChIP DNA and input controls were generated using the NEBNext Ultra DNA Library Prep Kit for Illumina (NE Biolabs) following the manufacturer’s protocol.