Anti c myc agarose

Isolation of a nuclear protein fraction and a Triton X-100–insoluble fraction (chromatin-bound fraction), immunoprecipitation, and immunoblot analyses were performed as previously described, with slight modifications [39 (link)]. For nuclear protein extraction, cell pellets were resuspended in MBS-A buffer (10 mM HEPES (pH 7.5), 340 mM sucrose, 1.5 mM MgCl₂, 10 mM KCl, 10%glycerol, 1 mM DTT, 0.1 M PMSF, phosphatase inhibitors and protease inhibitors) and incubated for 8 min at 4°C. After centrifugation, nuclei were further lysed in buffer X (100 mM Tris–HCl, 250 mM NaCl, 1mM EDTA, 1% NP-40, 0.1 M PMSF, phosphatase inhibitors, and protease inhibitors) with Benzonase nuclease followed by sonication and centrifugation. Anti-FLAG M2 agarose (A2220, Sigma), anti-c-Myc agarose (A7470, Sigma), anti-HA agarose (A2095, Sigma), anti-V5 agarose (A7345, Sigma), and anti-S-protein agarose (69704, Novagen) affinity beads were used for immunoprecipitation, as described previously. Agarose beads were incubated with cell lysates for 2 h at 4°C, washed three times with Buffer X at 4°C, and bound proteins were eluted with FLAG-peptide (19–73301, Peptron) or collected by denaturation with 2X SDS loading buffer and resolved by SDS-PAGE and immunoblotting.

Cells were lysed with cell lysis buffer (20 mmol/L HEPES KOH (pH 7.3), 150 mmol/L KCl, 10 mmol/L MgCl₂ and 1% NP40) (Heiman et al., 2014 (link)) supplemented with the protease inhibitor cocktail (Roche), 1 mmol/L DTT, 1000 U/mL RNase inhibitor, and 30 mmol/L NaBH₃CN (Sonenberg and Shatkin, 1977 (link)) for 30 min on a roller at 4°C. The lysate was clarified by centrifugation at 12,000 rpm for 15 min at 4°C. The clarified lysate was incubated with Anti-c-Myc Agarose (Sigma) and Anti-Flag M2 affinity gel (Sigma) separately for 2 h on a roller at 4°C. Immunoprecipitates were washed three times with TBST. Ten percents of the immunoprecipitate was resuspended in SDS loading buffer for protein detection by Western blotting. The remaining immunoprecipitate was resuspended in TRIzol (Invitrogen) for RNA extraction.

HEK293T cells were seeded in 10-cm dishes (3.5 × 10⁶ cells per dish) and transfected with the plasmids indicated in the Figs 1–7 using PEI. The following day, cells were washed twice with PSB, lysed in immunoprecipitation (IP) lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 0.5% (v/v) Nonidet P-40 (NP-40) and protease (cOmplete Mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche)) and cleared by centrifugation at 21,000 g for 15 min at 4°C. Cleared lysates were then incubated with 20 μL of ANTI-FLAG M2 Affinity Gel (Sigma Aldrich) or Anti-HA Agarose (Sigma Aldrich) for 2 h, or with 50 uL of Anti-c-Myc Agarose overnight, at 4°C. Alternatively, proteins were expressed by in vitro transcription/translation using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega) prior to incubation with the affinity resins. Immunoprecipitations were washed 3 or 4 times in IP buffer and bound proteins were eluted in Laemmli SDS-PAGE loading buffer and heated at 100°C for 5 min. Samples were then analysed by SDS-PAGE and immunoblotting.

The recruitment of KFL4 transcript to Ago2-complexes was determined as described [30 (link)]. Briefly, RAW264.7 cells were transfected with c-Myc-tagged Ago2 (a gift from Jidong Liu, Memorial Sloan-Kettering Cancer Center, NY) and miR-26a mimic or control mimic, allowed to grow for 24 h and lysed in cold lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, 100 units/ml RNasin Plus (Promega) supplemented with 1 mM PMSF and complete protease inhibitor cocktail (Roche) for 30 min at 4°C. Supernatants were incubated with anti-c-Myc-Agarose (Sigma) or protein A/G Plus-Agarose (Santa Cruz Biotechnology) that had been previously blocked in IP wash buffer (300 mM NaCl, 5 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 0.1% Triton X-100) and calibrated in lysis buffer containing yeast t-RNA (Promega) for 3 h at 4°C with rotation. Immune complexes were washed successively with lysis buffer, IP wash buffer and cold PBS to remove residual detergent. RNA was prepared using the miRvana kit (Ambion) according to the manufacturer’s instructions. RNA samples were eluted from columns in 30 μl H₂O and used for quantification of KLF4 by qRT-PCR. GAPDH was used for normalization.