Histone 3

In vivo UV-crosslinking and isolation of Arabidopsis RBPs was performed, as previously described [12 (link)], using a protocol that utilizes a modified method originally optimized for HeLa cells [11 (link)]. Sample from each time-point were split into two, one set for UV-crosslinking and the second set for non UV-crosslinking. Samples for UV-crosslinking were irradiated in vivo with UV (254 nm) and the mRNA-protein complexes were pulled down using oligo(dT) beads. Purified proteins were analyzed by label free tandem mass spectrometry. Similarly to [12 (link)], the quality of the mRNA-protein crosslinked complex pull-down was assessed by performing an additional control whereby the sample was treated with RNase T1/A mix (Thermo-Fisher Scientific) according to the manufacturer’s recommendations. To isolate RBPs, mRNA-protein samples were treated with RNase A/T1 mix to release them from the captured RNA molecules. Crosslinking and isolation of RBPs were evaluated by western blotting using antibodies against polypyrimidine tract-binding protein 1, β-actin (Sigma Aldrich, St Louis, MO, USA) and histone 3 (Abcam, Cambridge, UK) following manufacturer’s recommendations (see [12 (link)]).

Cells were plated onto 10 cm dishes and were treated as previously described. After 24 hours of incubating with Aβ, cells were collected and separated into nuclear and cytoplasmic fractions according to manufacturer recommended subcellular fractionation protocol (https://www.abcam.com/protocols/subcellular-fractionation-protocol). After protein concentration for all fractions were determined using a reducing agent-compatible BCA assay (ThermoFisher, cat no. 23252), 12.1 ug of protein from each sample was loaded onto a 10% Tris-Glycine gel (Biorad, cat no. 4561034) and then transferred onto a PVDF membrane and blocked for one hour with 5% BSA in 0.1% Tween/1xTBS. Membranes were probed with primary antibodies 4R tau (Millipore Sigma, cat no. 05–804), 3R tau (Millipore Sigma, cat no. 05–803), GAPDH (Abcam, cat no. 181602), and Histone 3 (Abcam, cat no. ab1791) overnight followed by the respective secondary HRP antibodies (mouse or rabbit) at 1:5000 and imaged using West Pico Supersignal (ThermoFisher, cat no. 34580).

Protein lysates of Vector‐TEVs, Foe‐TEVs or Foe‐Th cells were prepared according to standard procedures, and the contents of proteins were assessed by the BCA protein concentration measurement kit (Thermo Fisher Scientific). Subsequently, the samples were isolated using Bis‐Tris gel (Invitrogen), and then transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked in TBS‐T in 5% BSA for 1 h, incubated with primary antibodies overnight at 4 °C, then rinsing the membranes with PBS. Then, the membranes were covered with secondary antibody. Incubated at room temperature for 2 h, signals were assessed by enhanced Chemiluminescence Advanced System (GE Healthcare). The primary antibodies involving against CD9 (1:1000, Abcam), CD63 (1:1000, Abcam), Tubulin (1:1000, Abcam), TSG101(1:1000, Abcam), CTLA‐4 (1:1000, Abcam), TGF‐β (1:1000, Abcam), IL‐10 (1:1000, Abcam), PDCD‐1 (1:1000, Abcam), CD35 (1:1000, Abcam), CD73 (1:1000, Abcam), LAG3 (1:1000, Abcam), TIGIT (1:1000, Abcam), calnexin (1:1000, Abcam), histone 3 (1:1000, Abcam), and GM13 (1:1000, Abcam), ITGA4 (1:800, Abcam), ITGAL (1:800, Abcam), ITGB1 (1:800, Abcam), ITGB2 (1:800, Abcam) and secondary antibody Alexa Fluor Plus 800 (1:10 000, Thermos) were used.

15 µL protein sample was loaded into 10% polyacrylamide gel. Electrophoresis and transfer were performed using Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, USA). The membrane was blocked for 1 h with 5% BSA in TBST buffer (Tris: 20 mM, NaCl: 150 mM, Tween® 20 detergent: 0.1% (w/v)). Immunoblot analysis was conducted with the following antibodies: β-Actin (Sigma, A2228, 1:1000); H3K27Me3 (Abcam, ab6002, 1:1000); Histone 3 (Abcam, ab1791, 1:1000); anti-Brg1 (Abcam, ab4081, 1:1000); Ezh2 (Cell Signaling, D2C9 #5246, 1:1000); REST/NRSF (Millipore, 17-641, 1:1000); GAPDH‐HRP (Prosci, HRP‐60004, 1:1000); goat anti‐mouse IgG HRP (ThermoFisher Scientific, 62‐6520, 1:2500), and goat anti‐rabbit IgG HRP (ThermoFisher Scientific, 65‐6120, 1:2500). Protein bands were developed with Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, 32209) and were imaged using the ChemiDoc MP Imaging system (Bio‐Rad, Hercules, CA, USA). Relative band densities were analyzed using ImageJ.

Equal amounts of each protein diluted in 5 × sodium dodecyl sulfate (SDS) loading buffer (Beyotime, Shanghai, China) were separated using SDS–polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). After being blocked at room temperature for 1 h, the membranes were incubated with primary antibodies against GAPDH (1:1000, Cell Signaling Technology, Massachusetts, USA, 5174), p21 (1:1000, Abcam, Cambridge, UK, 109,520), p53 (1:1000, Cell Signaling Technology, 48,818), p16 (1:1000, Abcam, 51,243), SERPINE2 (1:1000, Abcam, 134,905), YBX3 (1:2000, Bethyl, Montgomery, USA, A303-070A), β-Tubulin (1:1000, Cell Signaling Technology, 2128), PCNA (1:1000, Abcam, 29), Histone3 (1:1000, Abcam, 176,842), ubiquitin (1:1000, Abcam, 134,953), HNRNPC (1:1000, Abcam, 133,607), YBX1 (1:1000, Proteintech, Rosemont, USA, 20,339–1-AP), and ZO-1 (1:1000, Proteintech, 21,773–1-AP) overnight at 4 °C. The membranes were washed 3 times, incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, BOSTER, California, USA, BA1050, BA1054) for 1 h at room temperature and then washed 3 times with TBST. Specific immunoreactive bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The mean intensity ratio was analyzed by ImageJ 1.4.

Rat Ter119-Alexa647 (Biolegend, #116218, 1:200), rabbit Nemp1 (1:1,000), mouse Lap2 (BD Biosciences, #611000, 1:200), Rat ckit-Alexa647 (#105817, 1:200), and goat Lamin B1 (Santa Cruz Biotechnology) were used for immunofluorescence microscopy. Fluotag-X4 anti-Rabbit IgG-Atto488 (Nanotag, #N2404, 1:500) and FluotagX2 anti-mouse IgG-AberioSTAR-580 (Nanotag, #N1202, 1:500) were used as secondary antibodies. Rat Ter-119-PE (Biolegend, #116208, 1:200) and Rat CD71-APC (Biolegend, #113820, 1:200) were used for FACS and flow imaging. Histone3 (Abcam, #1791, 1:2,000), Nemp1 (1:1,000), and Ter119-Biotin (Biolegend, #116204, 1:500) were used for immunoblotting.