Plant protease inhibitor mixture

C. reinhardtii strains, expressing FLAG-AGO3 or FLAG-Ble, were grown to midlogarithmic phase in TAP medium, and ∼4 × 10⁹ cells were pelleted by centrifugation and resuspended in 10 mL of lysis buffer (50 mM Tris⋅HCl, pH 7.5, 120 mM NaCl, 2 mM benzamidine, 0.2 mM PMSF, and 10% glycerol) containing 5 μL/mL plant protease inhibitor mixture (Sigma). From this step on, cells and lysates were always kept on ice. Cells were broken by 2 passages through a French press at a pressure of ∼2,000 psi. Cell lysates were then centrifuged at 16,000 × g for 30 min. CHAPS (0.1% final concentration) was added to the supernatant and an aliquot (∼10%) was saved for input control analyses of proteins and RNAs. The remaining supernatant was incubated with buffer-equilibrated anti–FLAG-M2 agarose beads (Sigma) for 2 h, and then the beads were washed 5 times with lysis buffer containing 0.1% CHAPS and 5 μL/mL plant protease inhibitor mixture. Proteins/RNAs associated with the beads were eluted by incubation with lysis buffer (without glycerol) containing 150 μg/mL 3×FLAG peptide (Sigma). RNA was purified from the eluate, precipitated with glycogen as carrier, and used for RT-PCR analyses (55 ).

An antisense LNA oligo pull-down assay was adapted from (55 ,56 (link)) with modifications. Two antisense LNA oligos were designed to target the accessible regions within AtTR and labeled with Biotin at the 3′ end. About 2 g of formaldehyde-crosslinked four-day-old WT (WS) or attr seedlings were homogenized in lysis buffer (50 mM Tris-OAC pH 7.5, 10 mM EDTA, 1% SDS, 20 μl/ml Plant protease inhibitor mixture [Sigma-Aldrich], 1 μl/ml RNaseOUT [Thermo Fisher Scientific] and 6 mM DTT). The pre-warmed 2× hybridization buffer (50 mM Tris-OAC pH 7.5, 750 mM NaCl, 15% formamide, 1 mM EDTA, 20 μl/ml Plant protease inhibitor mixture [Sigma-Aldrich], 1 μl/ml RNaseOUT [Thermo Fisher Scientific] and 6 mM DTT) and 100 pmol antisense LNA oligos were incubated at 42°C for 2 h to achieve oligo annealing. About 100 μl Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) was added and incubated for additional 45 min at 42°C. Beads were washed five times with wash buffer (2× SSC, 0.5% SDS, 1 mM PMSF, 5 mM DTT) before resuspending 90% of beads in SDS-loading buffer for protein analysis by western blot. The remaining 10% of beads was subjected to protease K digestion (10 mM Tris-Cl pH 7, 100 mM NaCl, 1 mM EDTA, 0.5% SDS and 5% 20 mg/ml protease K) at 50°C for 45 min prior to the RNA extraction by TRIzol reagent. The extracted RNA was analyzed by RT-qPCR for RNA abundance.

To purify VIG1-associated polypeptides, a complemented vig1 transgenic strain [vig1(tagVIG1)-3] was grown to midlogarithmic phase in TAP medium (containing 7 µM 5-FI) and cells were collected by centrifugation. For each experiment, ∼2 × 10¹⁰ cells were resuspended in lysis buffer (20 mM Tris⋅HCl, pH 7.5, 150 mM NaCl, 2 mM Mg[CH₃COO]₂⋅4H₂O, 2.5 mM CaCl₂, 1 mM imidazole, 10 mM β-mercaptoethanol, and 10% glycerol) supplemented with 2.0 mM benzamidine, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 5 µL/mL plant protease inhibitor mixture (Sigma), and 30 μg/mL RNase A. Cells were broken by 2 passages through a French press at 5,000 psi and the lysate was clarified by centrifugation at 16,000 × g for 30 min at 4 °C. The extract was then incubated with buffer-equilibrated calmodulin-Sepharose beads (GE Healthcare) for 16 h at 4 °C. Subsequent purification steps were performed as previously described (58 (link)). FLAG-CBP-Ble purification, following the same protocol, was used as a negative control. Isolated proteins were fractionated by 10% SDS/PAGE, stained with Sypro Ruby (Bio-Rad), digested in-gel with trypsin, and identified by tandem mass spectrometry as described (58 (link)). We observed that VIG1 migrates anomalously on SDS/PAGE and appears larger than its predicted size, as previously reported for mammalian SERBP1 (35 (link)).