Magnetic beads

RNA immunoprecipitation was carried out using embryo lysates as described (17 (link)). Embryos were collected at the 1 cell and 1000 cell stages, cross-linked using 1% formaldehyde and lysed in RIPA buffer (50 mM Tris–Cl pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, protease inhibitor cocktail). Anti-Ybx1 antibody (Sigma 4F12) was bound to 50 μl of magnetic beads (BioRad), incubated with 250 μl wild type embryo lysate at 4°C, washed with high stringency RIPA buffer (50 mM Tris–Cl pH 7.5, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1mM EDTA, 1 M NaCl, 1 M urea, protease inhibitor) and eluted with 100 μl of elution buffer (50 mM Tris–Cl pH 7, 5 mM EDTA, 10 mM DTT, 1% SDS) by heating at 70°C for 10 min. 40 μl of the eluate was used for western blot analysis and 60μl was used for RNA extraction using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using SuperScript(TM) III Reverse Transcriptase (Invitrogen), followed by RT-PCRs to detect sqt, lefty1, lefty2 and gapdh transcripts. The cycle numbers for the PCR reactions were: gapdh 25 cycles, sqt 28 cycles, lft1 29 cycles and lft2 28 cycles. The list of primers is included in the Supplementary material.

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 Tri1‐GFP or α₁‐3 × FLAG‐fusion constructs were verified by DNA sequencing and transformed into PH‐1 or corresponding mutants. Transformants expressing Tri1‐GFP and β₂ tubulin or Tri1‐GFP and α₁‐3 × FLAG were confirmed by western blotting analysis. In addition, the transformants expressing a single tag protein were used as references. For Co‐IP assays, magnetic beads (Bio‐Rad) were first incubated with the monoclonal anti‐GFP antibody 300943 following the manufacturer's protocol. Thereafter, the magnetic beads were incubated with total protein samples. Protein samples (10 μl) eluted from magnetic beads were analysed by western blotting with a polyclonal anti‐β₂ antibody IF11 (Zhou et al., 2016) or a polyclonal anti‐FLAG A9044 (Zenbio). Total protein samples were further assayed with monoclonal anti‐actin antibody 700068 as a reference. All experiments were repeated twice.

For immunoprecipitation, as described previously ^{58 (link)}, whole protein lysates (prepared from Capan-2 and AsPC-1 cells) were extracted in a lysis buffer (20 mM Tris/HCl, pH7.4, 1.0% NP-40, 1 mM EDTA, 150 mM NaCl, 50 μg/ml PMSF, 10 μg/ml leupeptin). Briefly, CRT, c-Myc and IgG (Santa Cruz) antibodies were preincubated with magnetic beads (Bio-Rad, Hercules, CA, USA) for 4 h at 4 °C. The antibody–beads complex was washed three times with the lysis buffer and incubated with the soluble supernatants of protein lysates overnight at 4 °C. Next, the immunocomplexes were washed three times with lysis buffer, eluted by boiling in sample loading buffer for SDS-PAGE and then subjected to WB analysis with CRT, c-Myc, Integrinβ1, Fibronectin and GAPDH antibodies. Each experiment was repeated three times.

The immunoprecipitation protocol used was described previously [28 (link)]. Briefly, the immunoprecipitation buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton, 1.5 mM MgCl2, 10% glycerol, EDTA-free Protease Inhibitor Cocktail (4693159001 Roche), PhosSTOP^TM (4906845001 Roche)) was used to lysate the cells. Cells were then centrifuged and supernatants were incubated at 4 °C overnight with magnetic beads (161–4021, Bio-Rad, Madrid, Spain) previously conjugated to 5 μg of rabbit anti-MCL-1 antibody (CST94296, Cell Signaling, Leiden, The Netherlands) or 5 μg of rabbit IgG antibody (CST2729, Cell Signaling). After magnetization, supernatant was discarded and the binding fraction was resuspended in 40 μL 4X SDS-PAGE sample buffer and heated at 70 °C for 10 min. Finally, the sample was magnetized to collect the supernatant, which was stored at −80 °C for further analysis.

Serum samples (n = 9–10 per group) were analyzed for levels of TNF-α (LLOD:1.4 pg ml⁻¹), IL-1β (LLOD: 9.4 pg ml⁻¹), IL-6 (LLOD: 0.2 pg ml⁻¹) and MCP-1 (LLOD: 3.7 pg ml⁻¹) by Bio-Plex immunoassays (assay range: 2–3000 pg ml⁻¹; intra-assay coefficient of variation: <10%; inter-assay coefficient of variation: <30%) formatted on magnetic beads (Bio-Rad Laboratories Inc, CA, USA), following the manufacturer’s instructions^{37 (link)}. Xponent software (Luminex, Austin, TX) was used for data acquisition and analysis. ELISA kits were used to analyze serum levels of sCD14 (LLOD: 0.06 ng ml⁻¹; inter- and intra-assay coefficients of variation were <12 and <8%, respectively) (MyBioSource, San Diego, CA) and LBP (LLOD: 0.4 ng ml⁻¹; coefficient of variation %: <6) (NeoBioLab, Cambridge, MA), according to the manufacturers’ instructions. For all the assays mentioned previously, 5–6 standards including blank (negative control) were used.

For patient P1, serum was diluted in sample buffer and assayed in multiplex on a Luminex Magpix system (Bio-Rad Laboratories, Hercules, Calif). Human IL-18BPa beads were generated with magnetic beads (Bio-Rad Laboratories) conjugated to clone MAB1192 and detected with clone BAF119 (both from R&D Systems, Minneapolis, Minn). Bioplex Pro group II cytokine standard was used for IL-18, whereas recombinant human IL-18BPa–Fc (R&D Systems) was used for IL-18BP. Patient P2's serum cytokine levels were quantified by using an ELISA for IL-1β (CHE001; 4A Biotech, Beijing, China) and IL-18 (CHE007; 4A Biotech), according to the manufacturer's guidelines.