Investigating Ubiquitination Signaling Pathways

Western blotting and IP assays were performed as described previously (25 (link), 26 (link), 28 (link)). Briefly, HEK293T cells were transiently transfected with mock as control vector, Flag-USP14, Myc-Beclin 1 wild type (wt), Myc-Beclin 1 truncated mutants, Flag-TRAF6 wt, Flag-TRAF6 truncated mutants, Myc-USP14 wt, HA-Ub, HA-TAB 2 wt, HA-TAB 2 truncated mutants, or Myc-Ub vector indicated in each experiment by using Lipofectamine 2000 (Invitrogen). At 38 h after transfection, transfected cells were extracted and immunoprecipitated with anti-Flag (Cell Signaling Technology, Beverly, MA, USA), anti-HA (Cell Signaling Technology), or anti-Myc antibody (Cell Signaling Technology). Immunoprecipitated complexes were separated by 6–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-HA, anti-Myc, or anti-Flag antibody. Ctrl and USP14^KD THP-1 cells treated with or without 3-MA (5 mM) and pepstatin A (10 µM) were stimulated with LPS (200 ng/ml) for 6 h. Whole cell lysates were subjected for immunoblot analysis of LC3A/B (4108, Cell Signaling Technology) and GAPDH (Cell Signaling Technology) as a loading control. Ctrl and USP14^KD THP-1 cells were treated with or without LPS (200 ng/ml) for different times. Cells were extracted, separated by 6–10% SDS-PAGE, and probed with the following antibodies: IκB-α, pho-p38, p38, pho-IKKαβ, IKKβ, and GAPDH purchased from Cell Signaling Technology (Beverley, MA, USA).