Ab106435

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.

Tumor tissues were immobilized with 4% formalin solution (Beyotime, Shanghai, China) and then paraffin-embedded. The 4 μm-thick sections were prepared. After blocking the endogenous peroxides and proteins and blocked with 5% goat serum, the sections were incubated overnight with the primary antibodies [including anti-Ki67 (ab16667, Abcam, MA, USA) and anti-THEM4 (ab106435, Abcam, MA, USA) monoclonal antibodies)] at 4°C. Then, the sections were cleaned with phosphate buffer saline (PBS), incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody at 37°C for 1 hour, and then stained with 3, 3-diaminobenzidine (DAB) solution for 3 min at room temperature. The nucleus was counted with hematoxylin. Finally, the staining was observed under Olympus BX 51 optical microscope (Olympus Corporation, Tokyo, Japan) at 200 × magnification.