PU-H71 induces ubiquitination and proteasomal degradation of STK33. (a) Immunoblot of A549 cells treated with 20 mM NH₄Cl for 2 h, followed by incubation with 0.5 µM PU-H71 for 16 h. One of two independent experiments is shown. (b) Immunoblot of A549 cells treated with 10 µM MG-132 for 2 h, followed by incubation with 0.5 µM PU-H71 for 16 h. (c) IP of HA-tagged STK33 from 293T cells treated with or without 1 µM PU-H71 for 7 h and 10 µM MG-132 for the final 3 h. Ubiquitinated STK33 appears as a smear with high molecular weight. One of three independent experiments is shown. (d) Anti-Flag IPs were performed with KRAS WT BT-20 and mutant KRAS-dependent MDA-MB-231 cells stably transduced with empty vector (EV), N-terminally Flag-tagged STK33 (Flag-STK33), or C-terminally Flag-tagged STK33 (STK33-Flag), and the resulting protein complexes were analyzed by mass spectrometry. The enrichment of peptides representing proteins involved in the ubiquitin/proteasome system is shown. (e) Immunoblot of MDA-MB-231 and A549 cells transduced with empty vector (EV) or BAG2 and treated with 0.5 µM PU-H71 for 24 h. (f) Immunoblot of HCT-116 and A549 cells transduced with a nontargeting control shRNA or shRNAs targeting BAG2. (g) Anti-Flag IPs were performed with MDA-MB-231 cells stably transduced with empty vector (EV) or Flag-tagged STK33, and immunoblots were probed with antibodies against BAG2 and Flag. One of two independent experiments is shown. (h) Immunoblot of 293T cells transfected with HA-tagged STK33 or Xpress-tagged USP15 and USP5, respectively, and treated with or without 0.5 µM PU-H71 for 24 h. One of two independent experiments is shown. (i) MDA-MB-231 cells were transfected with a nontargeting control shRNA or a shRNA targeting HERC2. After 3 d, cells were incubated with 1 µM PU-H71 for 18 h, as indicated. Protein levels were analyzed by Western blotting using the indicated antibodies. One of two independent experiments is shown.