Nupage novex 4 12 bis tris

Immunoblotting followed the same method done by Almughlliq, et al.^{25 (link)} to identify the exosomal fractions, which were then pooled accordingly. Exosomes (10 µg protein) were further characterized using gel electrophoresis (NuPAGE Novex 4–12% Bis-Tris, Thermo Fisher Scientific Australia Pty Ltd, Scoresby Vic) for confirmation of exosomal markers Flotillin 1 (FLOT1) and tumor susceptibility gene 101 (TSG101). The gel was then transferred to a PVDF membrane (Bio-Rad Laboratories Pty., Ltd, Australia) using Trans-Blot Turbo system (Bio-Rad Laboratories Pty., Ltd, Australia). After blocking (5% skim milk powder), membranes were probed overnight with primary antibodies anti-FLOT1 (1:500; Abcam, Cambridge, United Kingdom) and, anti-TSG101 (1:500; Abcam, Cambridge, United Kingdom) at 4 °C, followed by secondary anti-rabbit IgG (1:1000 Sigma–Aldrich, St Louis, MO, USA) and secondary anti-goat (1:1000; Sigma–Aldrich, St Louis, MO, USA), respectively. Membranes were then covered with SuperSignal West Dura-Extended Duration Substrate (Thermo Fisher Scientific Australia Pty Ltd, Scoresby Vic). Targeted proteins were visualized on X-ray films using Konica SRX101A processor (Konica Minolta medical and graphic INC, Japan).

Cell pellets were lysed in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors. Total protein concentrations were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For the synaptopodin, podocalyxin and aquaporin 1 Western blot, 10 μg total protein were loaded, while for the Cystinosin-3HA Western blot, 15 μg total protein was loaded on a precast gel NuPAGE Novex 4–12% Bis–Tris (Thermo Fisher Scientific, Waltham, MA, USA) and transferred on a nitrocellulose membrane using an iBlot2 dry blotting system.
Membranes were incubated overnight at 4 °C with primary antibodies against aquaporin 1, synaptopodin, podocalyxin and β-Actin on a blocking buffer. Next, membranes were incubated with HRP-linked secondary antibodies anti-rabbit or mouse in a 1:2000 dilution in 5% milk in TBS-T. Proteins were visualized using ECL Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired using the Syngene Chemi XRQ System and quantified with ImageJ software.
The specification of all antibodies is described in Table S3.

Cells were collected in dPBS by scraping and lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with protease (Merck Sigma) and phosphatase (Merck Sigma) inhibitors. Extracted proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Subsequently, 40 µg of protein was loaded on a precast gel NuPAGE Novex 4–12% Bis-Tris (Thermo Fisher Scientific). Proteins were then transferred onto a nitrocellulose membrane using an iBlot2 dry blotting system with iBlot2 transfer stacks (Thermo Fisher Scientific). Membranes were incubated for 1 h at room temperature in a blocking solution of 5% BSA in Tris buffer saline 0.05% Tween-20 (TBS-T). Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against γh2AX (1:2000) (Merck Millipore#05-636) and actin (1:5000) (Sigma-aldrich #A5441). The next day, membranes were incubated with HRP-linked anti-mouse secondary antibody (Cell Signaling Technology, 7076S) used at a dilution of 1:4000 in 5% milk in TBS-T. Bound antibodies were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Images were acquired using the ImageQuant LAS 4000 (GE Healthcare).