Anti rab10

Anti-B7-H3 (ThermoFisher, Loughborough, UK (IP), AF1027 BD Bioscience, Wokingham, UK (WB), and MAB1027 Santa Cruz, Heidelberg, Germany (IF)), anti-EEA1 (Cell Signaling, Leiden, The Netherlands), anti-HSC70 (Santa Cruz), anti-IMPDH2 (Proteintech), anti-Rab10 (Abcam, Cambridge, UK), anti-Rab11 (Cell Signaling), and HTII-280 AT2 (terrace Biotech, San Francisco, USA) were used. IgG negative control was used for IP and obtained from DAKO. DAPI (Sigma-Aldrich, Gillingham, UK) was used as nuclear stain for immunofluorescence. LysoTracker™ Deep Red was used to stain lysosomes (ThermoFisher). Anti-mouse-HRP, anti-rabbit-HRP, and anti-goat-HRP were from DAKO; anti-mouse-568, anti-rabbit-568, anti-rabbbit-488, and phalloidin-488 and -647 were all obtained from Invitrogen. Other reagents used were CellROX™ Deep Red Reagent (for oxidative stress detection) (ThermoFisher). CellEvent™ Caspase-3/7 Green Detection Reagent (ThermoFisher), Cisplatin (Cambridge Bioscience, Cambridge, UK), and Mycophenolic acid (MPA; IMPDH inhibitor) (Sigma-Aldrich).

Cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail and then total protein was collected for protein quantification through Pierce Bicinchoninic acid (BCA) Protein detection kit (Bio-Rad Laboratories, Hercules, CA, USA). Afterwards, 20 µg of protein was subjected to 12% SDS-PAGE, and then proteins were shifted to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). After being sealed with 5% BSA, the membranes were processed with primary antibodies at 4 °C overnight. The primary antibodies including anti-GAPDH, anti-cyclin D1, anti-CDK4, anti-caspase-3, anti-cleaved caspase-3, anti-caspase-6, anti-cleaved caspase-6, anti-PARP, anti-RAB10, anti-E6, anti-E7, were all procured from Abcam (Cambridge, MA). After washing in TBST, membranes were probed with HRP-conjugated secondary antibodies (Abcam) for 2 h at room temperature, and then protein signals were analyzed via the enhanced chemiluminescence substrate system (ECL; Santa Cruz Biotechnology, Santa Cruz, CA).