Magnetic bead

Palmitoylation of tissue protein was assessed by IP and acyl-biotin exchange (IP-ABE) as previously described [35 (link)]. Briefly, protein from the testis tissue was extracted using a lysis buffer with protease inhibitors and N-ethylmaleimide. After precipitating VMP1 protein with VMP1 antibody (#12929; Cell Signaling Technology; USA) and magnetic beads (Millipore), samples were re-suspended with stringent buffer. Each sample was divided into two parts: two thirds of the lysis sample part were mixed with 500 μl 1.5 M NH₂OH solution (Sigma-Aldrich) (+ NH₂OH Sample), and the one third part was mixed with 500 μl 0.1 M Tris–HCl (pH 7.4) (-NH₂OH Sample). All samples were rotated at room temperature for 50 min. Then samples were added with Biotin-BMCC buffer, and rotated for 50 min at 4°C. Proteins were eluted from the beads by boiling in sample buffer and the cleared supernatants were resolved by SDS-PAGE and electro-transferred to PVDF membranes, and then followed by western blotting.

Total protein was isolated from Col-0, pad2.1, and AtECS1. About 3 mg of total proteins was inoculated in protein isolation buffer containing protein A (magnetic beads, Millipore) and rabbit polyclonal anti-HSP70 primary antibody (Agrisera) overnight at 4 °C. After that the protein A solution was centrifuged and the pellet was taken. Extracted pellet was washed three times by dissolving in protein isolation buffer. Twenty microliters of protein loading dye (2% SDS, 100 mM Tris–HCl pH 7.5, 10% glycerol, 0.5 mM EDTA, 10 mM DTT) was added to the washed pellet and heated at 70 °C for 10 min. From the upper eluted liquid samples, western blotting was performed by using rabbit polyclonal anti-glutathione-S-transferase (GST) phi primary antibody (Agrisera). As a control the expression of HSP70 was also checked by using the same eluted sample.

DRIP was performed as described previously with some modifications [54 (link)]. Genomic DNA was extracted from the indicated strains and digested with Hind III (Neb), EcoR I, BsrG I, Xba I, and Ssp I. The digested DNA was purified and immunoprecipitated with S9.6 antibody (Millipore) in DNA binding buffer (10 mM Na₂HPO₄, 140 mM NaCl, and 0.05% Triton X-100) at 4 °C for 16 h. Protein A+G magnetic beads (Millipore) were used for recovering immunoprecipitated DNA and then washed 3 times with DNA binding buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, pH 8.0, and 0.5% SDS) and twice with TE buffer. The DNA was eluted from magnetic beads (Millipore) with elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, pH 8.0, and 0.5% SDS) and then treated with Proteinase K at 50 °C for 3 h. DNA was purified by phenol/chloroform/isoamyl extraction and then used for qPCR.

RIP assay was performed using the Magna RI RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. The magnetic beads (Bio-Rad, Hercules, CA, USA) were incubated with Argonaute-2 antibody (Anti-Ago2) or Immunoglobulin G antibody (Anti-IgG). Then, the HL-1 cell extract was incubated with RIP buffer containing magnetic beads conjugated to human anti-AGO2 antibody (Millipore, Billerica, MA, USA) and IgG control (Millipore, Billerica, MA, USA). The coprecipitated RNA was purified and quantified by qRT-PCR. RNA integrity was assessed with an Agilent 2100 Bioanalyzer.

The lysates of H1299 and A549 cells were collected and reacted with magnetic beads (Millipore) and anti-Ago2 or negative IgG antibody overnight at 4  C. After being mixed with proteinase K for 30 min, levels of molecules were tested by qRT-PCR [16 (link)].