Anti hsp90 b1

Protein samples were solubilized with a loading buffer (4×) (2-mercaptoethanol + NuPage; 1:5) and heated at 95 °C for 5 min for reducing conditions, and for non-reducing conditions, the loading buffer was used without 2-mercaptoethanol. Then, proteins were separated by SDS-PAGE using 10% acrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were incubated overnight at 4 °C with the primary antibodies anti-CD63 (mouse,1:1000, Clone Ts63, Invitrogen, Waltham, MA, USA), anti-ALIX (mouse,1:500, Clone 3A9, Invitrogen), anti-TSG101 (mouse,1:500, Clone 4A10, Invitrogen), anti-α-tubulin (mouse, 1:10,000, Clone DM1A, Invitrogen), and anti-HSP90-B1 (rabbit,1:4000, CUSABIO), followed by one hour of incubation at room temperature with ECL^TM Anti-mouse IgG and ECL^TM Anti-rabbit IgG, Horseradish Peroxidase linker (GE Healthcare, Chalfont St Giles, UK). The membranes were visualized with ECL^TM Prime Western blotting detection reagent (Amersham^TM) in the ChemiDoc Bio-Rad instrument (Hercules, CA, USA).

The primary antibodies: anti-Cyclophilin A (1:1000 dilution, Abcam, ab126738), anti-CD147 (1:1000, ThermoFisher, MA1–19,201), anti-HSP70 (1:3000, Santa Cruz, sc-66,048), anti-TSG101 (1:1000 dilution, Abcam, ab125011), anti-HSP90B1 (1:1000 dilution, Cusabio, CSB-PA10887A0Rb), anti-GAPDH (1:500 dilution, EMD Millipore, MAB374), anti-Rab7 (1:1000 dilution, Cell signaling Technology, 9367), anti-Rab11 (1:250 dilution, ThermoFisher, 71–5300).
The secondary antibodies: anti-Rabbit IgG-DyLight 800 (1:10,000 dilution, ThermoFisher, SA5–35,571), anti-Mouse IgG-DyLight 680 (1:10,000 dilution, ThermoFisher, 35,519)
Reagents: Trypsin (Pierce Trypsin Protease, MS Grade, ThermoFisher, 90,057), Propidium Iodide (ThermoFisher, P1304MP), 5-(and −6)-Carboxyfluorescein Diacetate, Succinimidyl Ester, mixed isomers (5(6)-CFDA, SE) (ThermoFisher, C1157)