Rabbit anti rab35

Cell cultures grown on coverslips were fixed with 4% PFA followed by permeabilization with 0.1% Triton X-100 and for 15 min at room temperature (RT) and then, blocked with 5% donkey serum for 1 h at RT. Primary antibody (rabbit anti-Rab35, 1:100; Abcam; rabbit anti-pser129 αSyn, 1:150; Abcam) diluted in antibody diluent (1% donkey serum + 0.1% Triton X-100) was incubated overnight at 4 °C. Secondary antibody (Goat-anti-rabbit Alexa 488, 1:500) was incubated for 1 h at RT. Cells were washed three times with PBS + 0.1% Tween-20. Coverslips were mounted on glass slides using VectaShield with DAPI mounting medium (Vector laboratories).
For whole brain organoids immunofluorescence, after being treated with 1 µM Alexa-633-tagged αSyn PFFs for 24 or 48 h, tissues were fixed in 4% PFA for 20 min at 4 °C followed by washing in PBS three times for 10 min before embedding in 4% low melting agarose (Sigma-Aldrich) in 1X PBS. Tissue sections of 50 µm were obtained using a vibratome (Leica VT1000 S Vibratome, Leica Microsystems, Wetzlar, Germany). For immunofluorescence, sections were blocked and permeabilized in 0.3% Triton X-100 and 4% FBS in PBS for 2 h at RT. Sections were then incubated with phalloidin Alexa-488 (Thermo Fisher, USA). Sections were washed three times with PBS + 0.1% Tween-20 and mounted on slides using VectaShield with DAPI mounting medium (Vector laboratories).

Western blot analysis was performed as described previously [5 (link)]. Briefly, proteins were separated using SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline with 5% milk and 0.05% Tween and incubated overnight at 4°C with primary antibodies including rabbit anti-Rab35 (1 : 1000, USA), rabbit anti-PI3K (Abcam), rabbit anti-Akt (Abcam), rabbit anti-mTOR (Abcam), rabbit anti-GFP (Abcam), and rabbit anti-PTPRN2 antibodies (Sigma). After being washed 3-5 times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Jackson ImmunoResearch) and visualized using enhanced chemiluminescence reagents.

Cell lysates were obtained by treating cells with RIPA buffer supplemented with inhibitors of proteases and phosphatases followed by centrifugation at 10,000 rpm for 10 min at 4 °C. 35 µg proteins were separated on 10% SDS–polyacrylamide or non-denaturing polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Millipore) and blocked in 3% (w/v) skimmed milk in Tris-buffered saline/0.1% Tween 20 (TBS-T). After, membranes were incubated over-night at 4ºC in primary antibody [(rabbit anti-alpha synuclein (Abcam), mouse β-actin (Santa Cruz) or rabbit anti-Rab35 (Abcam)] diluted in blocking solution. Membranes were washed with PBS and incubated with secondary antibody for 1 h at room temperature. The membranes were washed again in TBS-T and the signal visualized with ECL reagent (Millipore).