USP2A does not modulate the TRAF6-NF-κB pathway in HL-60 cells. (a, c, d, e) PMA (30 nM; 1 d)-pretreated USP2KD (KD) and control (Ctrl) cells and (b) USP2KD cells infected with USP2A (AR) or empty construct (mock) were stimulated with LPS (2.5 μg/mL) for (a–d) 1 h or (e) 0–2 h. Western blot analysis of TRAF6 protein levels in (a) USP2KD and (b) USP2A-restored cell. Values are normalized according to GAPDH levels. Data are presented as means ± SD of (a) nine or (b) seven samples. (c) K48- and K63-linked polyubiquitination of TRAF6 in USP2KD cells by immunoprecipitation-Western blot analysis. IP, antibody for immunoprecipitation; Det, antibody for detection. Arrowheads represent bands corresponding to the TRAF6 protein. Representative blot images of three independent experiments are shown. (d and e) Western blot analysis of nuclear RelA and p50 NF-κB and cytoplasmic IκBα in USP2KD cells. (d) NF-κB and IκBα protein levels are normalized to lamin-A and GAPDH levels, respectively. Data are presented as means ± SD of 16 (RelA and p50) or 12 (IκBα) samples. (e) Representative Western blot images of NF-κB components in the nucleus extracted from USP2KD and control cells after LPS stimulation.