Fig 1.

Hypoxia-induced VPS4B degradation. (A) Pa-4 cells were treated with desferrioxamine (DFO; 200 μM) for the indicated times in the presence or absence of MG132 (5 μM) during the last 6 h prior to harvesting. (B) SKBR3 and HEK293 cells were grown as either monolayer cultures (2D) or spheroids (3D) prior to hypoxia exposure. (C) Pa-4/TR/VPS4B(wt) and Pa-4/TR/VPS4B(EQ) cells were grown to form spheroids, which were exposed to 0.5% O₂ in the presence of bafilomycin A1 (Baf A1; 100 nM, 48 h) and MG132 (5 μM, last 5 h), as indicated. (D) HEK293 cells were transfected with Myc-VPS4B(wt) and HA-Ub, as indicated. At 48 h posttransfection, cells were exposed to 0.5% O₂ and treated with MG132 (5 μM, last 5 h of hypoxic exposure) prior to harvesting. Equal amounts of total cellular lysates were subjected to SDS-PAGE (A to C) or immunoprecipitation (D), followed by Western analysis with the indicated antibodies.