Ggpps

Cells or tissues were homogenised in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (ThermoFisher Scientific, USA). Equal amounts of protein lysate from each biological replicate were subjected to western blotting. Primary antibodies targeting CD63 (Abcam, 1:1000; ab68418), TSG101 (Abcam, 1:1000; ab125011), Syntenin-1 (Abcam, 1:1000; ab205861), Rab5 (Abcam, 1:1000; ab18211), Rab27A (Abcam, 1:1000; ab55667), CD81(Santa Cruz Biotechnology, 1:1000; sc-166029), Pgc1α (Santa Cruz Biotechnology, 1:1000; sc-518038), GGPPS (Santa Cruz Biotechnology, 1:200; sc-271680), β-actin (Santa Cruz Biotechnology, 1:1000; sc-47778), Calnexin (Cell Signaling Technology, 1:1000; 2433), CALR (Cell Signaling Technology, 1:1000; 12238), H3 (Cell Signaling Technology, 1:1000; 4499) and AGO2 (Proteintech, 1:500; 10686-1-AP) were used. Western blot data in the figures and supplemental figures are all representative of more than three independent experiments. The uncropped and unprocessed scans of the blots were provided in the Source Data file.

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.

Total proteins were isolated from tissues and cells, using lysis buffer containing mammalian protein extraction reagent RIPA (Beyotime, China), PMSF (Roche) and protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was determined using a Bio‐Rad Protein Assay kit (KeyGEN Biotech, Jiangsu, China). Protein samples (30 μg) were electrophoresed by 12% or 10% SDS‐PAGE, transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and incubated with specific antibodies (GGPPS, 1:500, sc‐271680, Santa Cruz Biotechnology, TX, USA; GAPDH, 1:10,000, ab181602, Abcam, Cambridge, UK; E‐Cadherin, 1:1000, 3195, Cell Signaling Technology; N‐Cadherin, 1:1000, 13116, Cell Signaling Technology; Vimentin, 1:1000, 5741, Cell Signaling Technology; Rac1/Cdc42,1:1000,4651, Cell Signaling Technology). After incubation with secondary antibody, protein bands were detected using an ECL detection system (Tanon, China).