Ab263019

Cells cultured in Matrigel were collected and lysed with RIPA buffer (MCE) containing protease and phosphatase inhibitors (MCE). Protein concentrations were determined by the BCA Protein Assay Kit (23227, Thermo Fisher Scientific). Western blotting was conducted according to a standard protocol. Briefly, the proteins were loaded on 10% SDS-polyacrylamide gels and electrophoretically transferred to a PVDF membrane (Millipore). The primary antibodies were purchased as follows: p-Akt (1:1000, ABclonal, AP0637), Akt (1:1000, ABclonal, A17909), p-GSK3β (1:1000, ABclonal, AP1088), GSK3β (1:1000, ABclonal, A6164), β-CATENIN (1:1000, ABclonal, A19657), β-actin (1:1000, ABclonal, AC026), anti-CD9 (1:1000, Abcam, ab263019), anti-CD81 (1:1000, Abcam, ab109201), anti-Hsp70 (1:1000, Abcam, ab181606), anti-TSG101 (1:1000, Abcam, ab125011), anti-Calnexin (1:1000, Abcam, ab133615), anti-CD63 (1:1000, Abcam, ab134045). Detection was performed using a Chemiluminescent Western Blot detection kit (4AW012-1000, 4A Biotech). The western blot results were analyzed using ImageJ (version 1.52). Uncropped and unprocessed scans of the blots could be obtained from the source data file.

The LoVo and SW480 cells or exosomes were collected, lysed with lysis solution (Roche) on ice for 30 min, and the total protein was separated by centrifugation at 14000 rpm for 30 min. Afterward, 50 μg total protein was loaded on 12% polyacrylamide gel and went through 2-hour electrophoresis at 100 V. The separated protein was then transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk at room temperature (RT) for 1 hour, the membranes were washed with TBST 3 times (10 min each time) and incubated with the antibodies (all from Abcam, MA, USA) of CD9 (ab263019, 1: 1000), CD63 (ab271286, 1: 1000), HSC70 (ab76005, 1: 1000), THEM4 (ab106435, 1:1000), p-AKT (ab38449, 1:1000), AKT (ab8805, 1:500), p-NF-κB (phospho S536) (ab106435, 1: 1000), NF-kB (ab32536, 1: 1000), and GAPDH (ab8245, 1: 1000) at 4°C overnight. After the membranes were rinsed with TBST, they were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody (concentration 1:300) for 1 hour at RT. Next, TBST was employed to wash the membranes 3 times (10 min each). At last, a Western blotting reagent (Invitrogen) was used for blots imaging, and the gray intensity of each protein was determined via Image J.

To extract total protein from plasma exosomes, exosomes were first exposed to Proteinase K treatment and were collected by Radio Immunoprecipitation Assay (RIPA) lysis buffer that contained protease inhibitor cocktail for a 30-min period on ice, followed by 10-min centrifugation at 13,000 × g and 4°C. Thereafter, this work adopted Pierce^® BCA Protein Assay kit (Thermo Scientific, USA) in quantifying exosomal proteins. For the detection of the exosomal biomarker proteins, exosome lysates (30 μg) were separated on 12% tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electronic blotting on the 0.2-μM nitrocellulose (NC) membrane (Millipore). Later, Western blot (WB) assay was conducted with anti-CD63 (1:1,000, abcam, #ab134045), anti-CD9 (1:1,000, abcam, #ab263019), and anti-CD81 (1:1,000, abcam, #ab109201) antibodies. The Gel Imaging System (Syngene G: BOX F3, USA) was employed to observe signals.

Proteins from serums or cells were isolated by RIPA buffer (Millipore) and quantified by a BCA protein assay kit (Tiangen, Beijing, China). Next, equal amounts of protein were separated via sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and then transferred onto polyvinylidene difluoride membranes (PVDF, Beyotime). After blocked with 5% defatted milk for 1 h at indoor temperature, the membranes were incubated with primary antibodies against OXSR1 (1:2000; ab97694; Abcam), CD9 (1:1000; ab263019; Abcam), CD63 (1:1000; ab134045; Abcam) and GAPDH (1:10,000; ab181602, Abcam) overnight at 4 °C. Then, the proteins on the membrane were probed with the secondary antibodies (1:20000; ab205718 or ab205719; Abcam) at 37 °C for 2 h. The protein blots were visualized using BeyoECL Star Kit (Beyotime).

Proteins from cells or exosomes were suspended in sodium dodecyl sulfate (SDS) loading buffer. After boiling, protein samples were separated on a 10% SDS-PAGE Gel and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Next, the membranes were incubated with primary antibodies against CD9 (ab263019, Abcam, Cambridge, UK), CD63 (ab134045, Abcam, Cambridge, UK), calnexin (ab133615, Abcam, Cambridge, UK), IL-1RA (ab124962, Abcam, Cambridge, UK), and β-Actin (TA-09, ZSGB-BIO, China) at 4 °C overnight, and then with secondary antibodies against rabbit (ZB-2301, ZSGB-BIO, China) or mouse (ZB-2305, ZSGB-BIO, China) for 1 h at RT. Finally, the signals were visualized.