Rap1a

Proteins derived from cells and EVs were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto nitrocellulose membranes, bound with primary antibodies, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence detection reagents (34580, Thermo Scientific, Waltham, MA, USA). Primary antibodies against the following proteins were used: Alix (ab56932, Abcam, Cambridge, UK), CD63 (ab68418, Abcam), TSG101 (ab56932, Abcam), Synthenin-1 (ab133267, Abcam), β-actin (112620, Cell Signaling Technology, Danvers, MA, USA), PD-L1 (13684, Cell Signaling Technology), Rab27a (ab55667, Abcam), Rab11 (ab18211, Abcam), Rab7 (ab50533, Abcam), pan Ras (sc-166691, Santa Cruz, Santa Cruz, CA, USA), Rap1A (sc-398755, Santa Cruz), p-B-Raf (2696S, Cell Signaling Technology), B-Raf (9433S, Cell Signaling Technology), p-MEK (9154S, Cell Signaling Technology), MEK (9126S, Cell Signaling Technology), p-ERK1/2 (4370S, Cell Signaling Technology), ERK1/2 (4695S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), and AKT (9272S, Cell Signaling Technology).

The cerebellum was dissected from each mouse. Tissues were homogenized in cold radio-immunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Thermo). Lysates were cleared by centrifugation (12,000 rpm for 20 min). The concentration of protein samples was determined using a standard BCA Protein Assay (Thermo). Loading protein samples were mixed with a loading buffer and boiled at 99 °C for 5 min. 30 μg total protein samples were resolved in a 10% or 12% SDS–PAGE for electrophoresis. Proteins in an electrophoresis gel were transferred to polyvinylidene fluoride membranes (Roche). The membrane was blocked with 5% non-fat milk (w/v) for 1 h and incubated with one of the following primary antibodies overnight at 4 °C: Ggpps (Santa Cruz, sc-271680), Pax6 (Biolegend, 901301), Rap1A (Santa Cruz, sc-65), Rap1 (Santa Cruz, sc-398755), RhoA (Santa Cruz, sc-418), p21 (CST, 64016S), Atp1a1 (Proteintech, 55187-1-AP), β-actin (ABclonal, AC026). After it was washed out for three times, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The membrane was visualized using an enhanced chemiluminescence system (Tanon-4600, Tanon, China). ImageJ was used to quantify intensities for targeted protein bands.