Bolt 4 12 bis tris plus gel

Cells were lysed with Cell Lysis Buffer (Cell Signaling Technology) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). For Western blot to confirm expression of membrane-localized CD4 fusion protein in Fig. 5B, cells were lysed in RIPA buffer made using recipe found here: https://www.abcam.com/protocols/general-western-blot-protocol#Solutions%20and%20reagents. Lysates were mixed with NuPAGE LDS Sample Buffer (Life Technologies) and NuPAGE Sample Reducing Agent (Life Technologies) and were run on Bolt 4–12% Bis-Tris Plus gels (Invitrogen). Gels were transferred with iBlot2 PVDF Mini Stacks (Invitrogen) and iBlot2 (Invitrogen). Blots were probed with antibodies for mCherry (GTX128508, GeneTex), GFP (sc-99G, Santa Cruz Biotechnology), Tid1 (EPR12414, abcam or RS-11, Santa Cruz Biotechnology), p53 (DO-1, Santa Cruz Biotechnology), CHOP (9C8, Novus Biologicals), ATF6-N (70B1413.1, Novus Biologicals), pEIF2a (9721, Cell Signaling Technology), EIF2a (9722, Cell Signaling Technology), Bax (6A7, Santa Cruz Biotechnology), MDM2 (sc-965, Santa Cruz Biotechnology), Cyclophillin D (E11AE12BD4, abcam), Hsp90 (C45G5, Cell Signaling Technology), Cox IV (3E11, Cell Signaling Technology), and GAPDH (D4C6R, Cell Signaling Technology). Blots were imaged using Bio-Rad ChemiDoc MP Imaging System or Azure Biosystems Sapphire Biomolecular Imager.

NGN3 + /IPC, NGN3^Lo, and EP stage cells were dissolved in RIPA buffer (1 × 10⁶ cells/ml, NACARAI TESQUE). Cell lysate were loaded on the Bolt 4–12 Bis-Tris Plus gels (INVITROGEN) and transferred to PVDF membrane by iBlot2 Transfer Stacks (INVITROGEN). After that, membrane blocking for 1 h in 5% Dry milk/PBS. Then, membrane was incubated with primary antibody against human NGN3(SANTACRUZ, #sc-376607) and human GAPDH (CALBIOCHEM, CB1001). After washing the membrane, membrane was incubated with second antibody against anti-mouse IgG (KPL, 474-1806), and Signals were then prepared by ECM solution (immobilon, MILLIPORE). Chemiluminescent Signals were detected by the Chemidoc Touch Imaging system (BIO-RAD).

BCC cells (3 × 10⁶) were lysed in ice-cold cell extraction buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 20 μL/mL protease inhibitor cocktail (Sigma-Aldrich, Germany) and 1 mM PMSF (Abcam, Cambridge, U.K.) for 30 min. The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. Qubit® protein assay kit (Invitrogen) was used to determine total protein concentration by Qubit 3.0 fluorometer (Invitrogen). Cell lysates (30 μg) were separated by Bolt™ 4–12% Bis-Tris plus gels (Invitrogen) in MES SDS running buffer and transferred to 0.45 μm PVDF membranes (GE Healthcare, U.K.). Proteins were detected using primary antibodies against ECHS1 (ab174312) and FASN (ab99359) (Abcam), PPAR-δ (PA1-823A), ME1 (MA5-23524), ME2 (PA5-38007), ME3 (PA5-36494), and GAPDH (AM4300) (Thermo Fisher Scientific, USA), and a WesternBreeze® chemiluminescent kit (Invitrogen) according to the manufacturer’s instructions. Bands were visualized by G:Box Chemi Gel Documentation system (Syngene, Frederick, MD, USA).

Fifty micrograms of proteins from each experimental group were applied to Bolt 4–12% Bis-Tris Plus gels (Invitrogen, Karlsruhe, Germany) and electrophoresed for 2 h 30 min at 80 V. Proteins were transferred onto a PVDF membrane in blotting buffer for 1 h at 100 V and blocked with 5% skim milk (Difco, Detroit, MI, USA) or 5% BSA (Gibco, Grand Island, NY, USA) in TBST for 1 h at room temperature. The blotted membrane was then incubated overnight at 4 °C with the different primary antibodies. Antibodies against GMPPA (1:4,000) and RRM2 (1:5,000) were purchased from Young in Frontier (Seoul, Korea), MAVS (1:5,000) was from Bethyl Lab (Montgomery, TX, USA), SOD1 (1:1000) and IPO4 (1:1000) were from Invitrogen (San Diego, CA, USA) and β-actin (1:10,000) was from Cell Signaling Technology (Beverley, MA, USA). Blots were then incubated with horseradish-peroxidase conjugated anti-rabbit IgG (GeneTex, Irvine, CA, USA, diluted 1:7,000 for GMPPA, 1:5,000 for MAVS and IPO4, Jackson ImmunoResearch, West Grove, PA, USA, diluted 1:10,000 for SOD1) and anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA, diluted 1:11,000 for RRM2) for 1 h at room temperature. Detection was performed using an ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Each tEV and nEV sample isolated from the serum was lysed using M-PER and Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of the exosome lysate was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Next, 20 μg of exosome lysates were separated on Bolt™ 4–12% Bis-Tris Plus Gels (Invitrogen, Carlsbad, CA, USA) and transferred onto PVDF membranes (Invitrogen, Carlsbad, CA, USA). The membranes were blocked with TBS-T supplemented with 5% skim milk for 1 h at RT. After blocking, the membranes were incubated at 4 °C overnight with the primary antibodies tumor susceptibility gene 101 (TSG101; Novus Bio, Littleton, CO, USA), CD171, β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), neuron-specific class III β-tubulin (TUJ1), neuronal nuclei (NeuN), and neuron-specific enolase (NSE) (Abcam, Cambridge, UK) (1:1000). After washing, the membranes were incubated with HRP-conjugated secondary antibody (1:2000) for 1 h at RT and washed with TBS-T. Finally, the protein bands were detected with EzWestLumi Plus (ATTO, Tokyo, Japan) and analyzed using ImageQuant™ LAS 4000 (GE Healthcare, Buckinghamshire, UK).