Running buffer

The group 1 mGluR agonist DHPG (50 μM, Tocris, UK) was used to activate group 1 mGluR. To block mGluR5 and mGluR1a, MPEP (10 μM, Tocris, UK) and LY367385 (100 μM, Tocris, UK) was applied to rat hippocampal slices, respectively. AM251 (3 μM, Tocris, UK) was used to block presynaptic CB1R. U73122 (5 μM, Tocris, UK) was used to block PLCβ activity. m-3M3FBS (30 μM, Tocris, UK) was used as the PLC activator. The NMDAR antagonist D-AP5 (50 μM, Tocris, UK) and the AMPA receptor antagonist CNQX (20 μM, Tocris, UK) were used for the eIPSC recordings. AβO and scrambled AβO were synthesized from a lyophilized powder of Aβ and scrambled Aβ peptide, respectively (Bachem, Japan). A 4× Laemmli sample buffer (Bio-Rad, USA) and running buffer (Bio-Rad, USA) were used for western blot SDS-PAGE. For the antibody incubation step in western blotting, rabbit monoclonal primary antibodies (NBP2-38220, Novus, USA) and HRP-conjugated secondary anti-rabbit antibodies (Cat# 170-6515, Control# 64170140, RRID: AB_2617112, Bio-Rad, USA) were used, respectively.

Cell lysates were prepared in RIPA buffer and 1× protease inhibitor cocktail (Roche Applied Bioscience, Penzberg, Germany). The proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN precast gels using a running buffer (Bio-Rad, Hercules, CA, USA) and were then transferred to PVDF membranes (0.45 μM). After being blocked with 5% nonfat milk, the membranes were incubated with the following primary antibodies according to the manufacturer’s recommendations: anti-cleaved BAX, BCL-2, caspase-3, phosphorylated I-kappa-B-alpha (p-IκBα), I-kappa-B-alpha (IκBα), phosphorylated p65 (p-p65), p65, actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-MALT1, K48-linkage-specific polyubiquitin, and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). The membranes were subsequently incubated with HRP-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA). To scan and analyze the membranes, an ImageQuant LAS 4000 mini imager (Fujifilm, Tokyo, Japan) and an image analysis program (Multi Gauge Ver. 3.0, Fujifilm) were used.

Cells were plated at 100,000 cells/well in 6-well plates and were allowed to adhere overnight prior to treating with 5 mM DFMO in RPMI-1640 with 10% FBS for 48, 72, or 96 hours. Drug supplemented medium was refreshed after 48 hours for 72 and 96 hours timepoints. H₂O was used as vehicle control. Cells were lysed with RIPA lysis buffer (Cell Signaling, Danvers, MA), supplemented with a complete protease inhibitor (Roche, Madison, WI). Cell lysates were collected and frozen at −80°C overnight to ensure complete cell lysis. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Lysates were electrophoresed on a 12% SDS-polyacrylamide gel in running buffer (Bio-Rad). Thirty μg of protein was loaded per lane. Gels were semi-dry transferred to nitrocellulose membranes using the Turbo Transfer system with Turbo Transfer buffer (Bio-Rad). Primary antibodies used were: rabbit polyclonal LIN28B, rabbit polyclonal MYCN, and rabbit polyclonal β-actin (Cell Signaling). Goat anti-rabbit secondary antibody was used (Licor). Protein bands were detected by fluorescence using the Odyssey infrared imaging system (Licor).

Western blotting was performed as described in Sambrook’s protocols [49 ]. Briefly, exosomes were treated with radioimmunoprecipitation assay (RIPA) lysis buffer at a ratio of 1:2 and incubated at 4 °C for one hour. The lysates were centrifuged at 18,000× g for 15 min at 4 °C, and the protein supernatant was collected. Bradford assay was performed for protein quantification, as mentioned earlier. The proteins were denatured using Laemmli buffer at 95 °C for 10 min. The sample was loaded on 10% gel in a running buffer at 120 V (BioRad system). The protein-containing gels were then wet transferred onto a nitrocellulose membrane at 350 mA for 100 min. The membranes were blocked with 5% skimmed milk in PBST (w/v) (0.1% Tween-20 in PBS (v/v)). An Abcam exosome antibodies panel containing CD9, CD63, CD81, TSG101, Hsp70, and Calnexin was used for the primary antibodies (Cat# ab275018: Exosome Panel), whereas secondary antihuman mouse antibodies were used at 1:5000 dilution. For MUC1 expression, the blots were incubated in primary MUC1 antibody (MUC1 VU4H5) at a dilution of 1:500. All antibodies were diluted in 2% skimmed milk in PBST (w/v). After membrane blocking, samples were washed three times with PBST before every step. After incubation with ECL for 2.0 min in the dark at room temperature, the membranes were imaged using the BioRad Chemidoc imaging system.

Cell lysates derived from antibody-treated cell lines and MPN was mixed with an equal volume of Laemmli buffer (Bio-Rad, CA, USA). The solution was vortexed then heated for 5 min to 95°C. The solution was left to cool down before loading the sample into 12% SDS-PAGE gel (Bio-Rad, CA, USA) and run at 200 Volt for 5 min then 1h 30 min at 100V in running buffer (Bio-Rad, CA, USA). Following transfer at 18V for 2h 30 min in transfer buffer (Bio-Rad, CA, USA), the membranes were blocked using 2% bovine serum albumin (BSA) (Sigma-Aldrich, USA) or 5% skimmed milk followed by human TrueStain FC_x™ blocker (Biolegend, San Diego, USA) (5µl/blot). The blots were rinsed with TBST and 0.5 µg/ml of primary antibody mouse anti-human FcϵRIα (Biolegend, San Diego, USA) was added for overnight incubation before washing with 0.1% TBST buffer. The secondary antibody goat anti-mouse IgG (Fab specific) (1:80000) (Sigma-Aldrich, USA) was then added for 1hour at room temperature. The blot was washed using 0.1% TBST then visualized using the Clarity Western ECL Substrate (Bio-Rad, CA, USA) in iBright™ CL1000 Imaging System (Thermo Fisher Scientific).