Fl 335

For overexpression experiments, GP130-deficient HEK293T cells were cotransfected with a WT or mutant pCMV6-GP130 vector or an empty pCMV6 vector and cultured for 24 h before harvesting and cell lysis. Total protein extracts were prepared by mixing cells with lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 0.5% Triton X-100) and incubating for 30 min at 4°C. The cells were centrifuged for 10 min at 16,000 ×g, and the supernatant was collected for immunoblotting. A mixture of protease and phosphatase inhibitors was added to the buffers immediately before use: aprotinin (10 µg/ml; Sigma-Aldrich), PMSF (1 mM; Sigma-Aldrich), leupeptin (10 µg/ml; Sigma-Aldrich), protease inhibitor cocktail (1×; Sigma-Aldrich). DNA was digested with Pierce Universal Nuclease for Cell Lysis (1/100; Thermo Fisher Scientific) during the lysis step. When indicated, PNGase F treatment was used in accordance with the manufacturer’s instructions (New England Biolabs) to eliminate N-glycosylation. The proteins were separated by SDS-PAGE and immunoblotting was performed with antibodies against GP130 (E-8; Santa Cruz) and GAPDH (FL335; Santa Cruz).

sEVs or cell lysates (10ug) were separated on 7–15% SDS/PAGE gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA) for western blot analysis. Membranes were incubated overnight at 4°C with antibodies specific for: CD34 (1:2000, ab81289, Abcam), CD81 (1:200, PA5-13,582, Thermo Fisher), HMGCR (1:2000, ab174830, Abcam), SREBP-2 (1:500, sc-271,616, Santa Cruz), LDLR (1:500, sc-18,823, Santa Cruz), GAPDH (1:500, FL-335, Santa Cruz), CD123 (1:1000, AF841,R&D), CLL-1 (1:2000, AF2946, R&D), β-Actin (1:500, sc-47,778, Santa Cruz), TSG101 (1:500, PA5-31,260, Thermo Fisher), ApoB (1:2000, 20,578-1-AP, Thermo Fisher), Calnexin (1:1000, #2433, Cell Signalling), Grp94 (1:1000, #20,292, Cell Signalling). Next, the HRP-conjugated secondary antibody (1:10,000, Pierce, Thermo Fisher) was added for 1 h at room temperature (RT) and blots were developed with ECL detection reagents (GE Healthcare Biosciences, Pittsburgh, PA, USA). The intensities of the bands on exposed films were quantified using Image J software (NIH, USA).

Protein was extracted from the six matched pairs of colorectal adenocarcinoms and corresponding paracancerous tissues with the RIPA lysis buffer containing PMSF protease inhibitors and was quantified by using the BCA assay (Beyotime Institute of Biotechnology, China). Samples of 50 μg of protein were loaded and resolved by SDS-PAGE followed by transfer to a PVDF membrane. Non-specific binding was blocked using 5% bovine serum albumin in Tris Buffered Saline with Tween 20 (TBS-T, pH 7.6) for 2 h. Subsequently, incubation with primary antibodies, rabbit polyclonal anti-PGAM5 (1:1000 dilution, ab126534; Abcam, United Kingdom), rabbit polyclonal anti-Parkin (1:1000 dilution, ab233434; Abcam, United Kingdom), and glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, 1:1000 dilution, FL-335; Santa Cruz Biotechnology, CA, United States) was undertaken overnight at 4 °C. After extensive washing, the membranes were incubated with anti-rabbit IgG secondary antibody (1:5000) and the immunoreactive bands were detected with ECL-Plus chemiluminescent detection HRP reagents (Beyotime Institute of Biotechnology, China) using the Microchemi 4.2 Bio-imaging system. The Western blots were quantified by densitometric analysis using ImageJ software (Version 1.46r). The experiments were repeated three times under the same experimental conditions.

The following antibodies were used for IB, IF, and IP experiments: anti-Drg1 antibody (dilution 1:1,000 for IB, PAB20044; Abnova), anti-Drg2 antibody (1:1,000 for IB, NBP2-16227; Novus Biological), anti-acetylated tubulin antibody (1:1,000 for IF, T7451; Sigma-Aldrich), monoclonal anti–α-tubulin antibody (1:500 for IF, T9026; Sigma-Aldrich), anti-Dvl2 antibody (1:1000 for IB, 1:200 for IP, 3216S; Cell Signaling Technology), anti-Dvl3 antibody (1:2,000 for IB, 2 µg for IP, 13444–1-AP; Proteintech), anti-phospho-cofilin (Ser3) polyclonal antibody (1:500 for IF, 44-1072G; Thermo Fisher Scientific), anti-phospho LIMK1 (phospho T508) antibody (1:500 for IF, ab38508; Abcam), anti-GAPDH antibody (1:5,000 for IB, FL-335; Santa Cruz Biotechnology), anti-GFP antibody (1 µl/tube for IP, 1:500 for IF, NB600-308; Novus Biological), anti-GST antibody (1:2,000 for IB, ab19256; Abcam), HA-tag (C29F4) antibody (1:1,000 for IF, 3724; Cell Signaling Technology), anti-V5-peroxidase antibody (dilution 1:10,000 for IB, V2260; Sigma-Aldrich), and monoclonal anti-Flag-M2 HRP antibody (dilution 1:5,000 for IB, A8592; Sigma-Aldrich).