Laemmli sample buffer

The RiboTag method was performed as described previously (65 (link)) with modifications. Briefly, E17.5 spinal cords were homogenized using TissueRuptor (Qiagen) in a buffer [50 mM tris-HCl (pH 7.4), 100 mM KCl, 12 mM MgCl_2, and 1% NP-40] supplemented with cycloheximide (0.1 mg/ml), heparin (1 mg/ml), SuperaseIn RNase (Thermo Fisher Scientific). Lysates were cleared by centrifugation at 10,000g for 10 min, and 5% of the lysate was saved as input. To reduce nonspecific-binding protein G magnetic beads (Dynabeads, Thermo Fisher Scientific) were added to the lysate and incubated for 2 hours at 4°C. Next, 5 μl of anti-HA antibody (Sigma-Aldrich) was added to the precleared lysate and incubated for 2 hours at 4°C. Later, 100 μl of beads slurry with 2 μl of SuperaseIn were added to the lysate followed by 2-hour incubation in the cold room. After precipitation, beads were washed five times in the wash buffer [300 mM KCl, 1% NP-40, 50 mM tris-HCl (pH 7.4), 12 mM MgCl₂, and cycloheximide (0.1 mg/ml)]. Beads were eluted in Qiazol (Qiagen), and RNAs were isolated using the RNeasy Micro Kit (Qiagen). Ten percent of the beads were used for WB and eluted in Laemmli sample buffer (Thermo Fisher Scientific) supplemented with 100 mM DTT. The quality control of RNA was performed using TapeStation (Agilent).

Cells were lysed in RIPA buffer (no. 89900; Thermo Fisher Scientific) supplemented with protease and phosphotase inhibitors (no. 87785; Thermo Fisher Scientific) and total protein extracts were boiled in Laemmli sample buffer (no. NP0007; Thermo Fisher Scientific), separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose using the iBlot2 system (Thermo Fisher Scientific). The membrane was blocked using blocking reagent (no. 11520709001; Roche Diagnostics) for 1 hour at room temperature and then incubated with a primary antibody against CXADR (no. AF3336; goat anti-hCAR; R&D Systems) overnight at 4°C. Subsequently, the membrane was incubated with a horseradish peroxidase–conjugated donkey anti-goat anti–immunoglobulin G secondary antibody (no. PA1-28664; Thermo Fisher Scientific) for 1 hour at room temperature. Immunoreactive bands were visualized by means of chemiluminescence (ChemiDoc XRS+; Image Lab Software; Bio-Rad). Blots were checked for equal loading by probing with a rabbit polyclonal anti-calnexin antibody [14 (link)].

Western blotting was performed as described previously^{72 (link)}. Briefly, cells were lysed for 30 min in RIPA buffer containing 1X protease inhibitor (Fisher Scientific, cat no. PIA32955) and centrifuged for 10 min at 14,800 rpm at 4 °C. Protein concentration for cell lysates and EVs were measured using the BCA protein assay kit (Life Technologies, cat no. 23225). EV lysates (4 μg/lane) and cell lysates (4 μg/lane and 25 μg/lane) were diluted in 1X Laemmli sample buffer (Fischer Scientific, cat no. BP-110NR-25ML) and were resolved on a 12% Polyacrilamide Gel (Bio-rad, cat no. 567-1043). The gel was transferred to a PVDF membrane (Fisher Scientific, cat no. IPFL00010) and subsequently blocked with Odyssey Blocking buffer (Licor, cat no. 927-40003) for 1 h at room temperature. Blocked membranes were incubated overnight at 4 °C with the following primary antibodies; anti-CD9 (1:1000), anti-CD81 (1:250, non-reducing conditions), anti-Grp94/GP96 (1:1000) and anti-β-actin (1:1000). Membranes were washed four times with 1X PBST and incubated for 1 h at room temperature with the following secondary antibodies: Licor IRDye 680RD Goat anti-Mouse IgG (1:10,000) or Licor IRDye 800CW Goat anti-rabbit IgG (1:10,000). After incubation with secondary antibodies, membranes were washed four times with 1X PBST and three times with 1X PBS, and signal was visualized with Li-Cor Odyssey CLX.

Cells were seeded at a density of 2.0 × 10⁵ cells/mL for 24 h prior to chemotoxic treatment. Cells were lysed as explained above. An amount of 4× Laemmli sample buffer containing β-mercaptoethanol (ThermoScientific, Waltham, MA, USA) was added to each sample and denatured at 95 °C for 10 min. An SDS polyacrylamide gel electrophoresis at 150 V for 45 min was utilized to resolve all proteins and transferred on a PVDF membrane using a TransBlot Turbo semi-dry transfer system (BioRad Laboratories, Hercules, CA, USA) for 7 min at 1.3 A and 25 V. Five percent milk w/v nonfat milk in phosphate-buffered saline + tween (PBST) was used as a blocking agent for 1 h post transfer. Primary antibodies were incubated overnight at 4 °C. Blots were then washed with 1× PBS supplemented with 0.01% tween. All secondary antibodies were incubated at room temperature for 1 h, and PBST washes were repeated. All Western blots were exposed via ECL reagent (Pierce, Waltham, MA, USA). A BioRad ChemiDoc MP imaging system was utilized to image all Western blots. The original western blot figures could be found in Supplementary File S1.