K63ub

Antibodies against USP7 (Cat. CST#4833), K48Ub (Cat. CST#4289), and K63Ub (Cat. CST#12930) were purchased from Cell Signaling Technologies, Inc. The antibodies against RNF6 (Cat. #20437-1-AP), USP9x (Cat. #55054-1-AP), PARP (Cat. #13371-1-AP), Caspase 3 (Cat. #19677-1-AP), GAPDH (Cat. #60004-1-Ig), α-tubulin (Cat. #11224-1-AP), and V5 (Cat. #14440-1-AP) were obtained from Proteintech. The monoclonal antibodies including anti-Flag (Cat. #M185-3L), anti-HA (Cat. #M180-3), and anti-Myc (Cat. #M192-3) were obtained from Medical and Biological Laboratories Co Ltd. The anti-Ub antibody (Cat. #SL-8017) was purchased from Santa Cruz Biotechnology, Inc. HRP-labeled goat anti-mouse (Cat. #A0216) and goat anti-rabbit IgG (H + L) antibodies (Cat. #A0208) were purchased from Beyotime Institute of Biotechnology. MG132 (Cat. #S2619), Bortezomib (Cat. #S1013), and P5091 (Cat. #S7132) were purchased from Santa Cruz Biotechnology and Selleck Chemicals Inc, respectively. CHX (Cat. #C7698) and chloroquine (CHQ, Cat. #C6628) were purchased from Sigma-Aldrich. LBH589 (LBH, Cat. #M1748) was purchased from AbMole Bioscience Inc. Nilotinib (Cat. #IN0560) was purchased from Solarbio Life Science. Recombinant ubiquitin (Cat. #U100-H), UBE1 (Cat. #E-304), UBE2D1(Cat. #E2-616), and ATP (Cat. #B-20) were all purchased from Boston Biochem Inc.

Immunoblotting and immunoprecipitation were performed as described previously (17 (link), 18 (link)). Briefly, all cells were harvested on ice using lysis buffer. N-ethylmaleimide (10 mM) was added to the lysis buffer when protein Ubiquitination was detected. Immunoprecipitation was performed using specific antibodies overnight on a rotor at 4°C. Equivalent quantity of proteins were subjected to SDS-PAGE followed by transferring to PVDF membranes (Millipore). Then membranes were blocked with 5% non-fat milk or 5% BSA, and incubated with the corresponding primary antibodies, followed by the respective HRP-conjugated Goat anti-mouse or Goat anti-rabbit (Bioworld) secondary antibodies. Immunoreactive bands were depicted using SuperSignal West Dura Extended kits (Thermo Scientific). The following antibodies specific for GST (1:2000, HaiGene, M0301), PKD1 (1:500, Santa Cruz, sc-639), β-TrCP (1:1000, Cell signaling, #4394), HA (1:2000, abcam, ab9110), K63Ub (1:500, Cell signaling, D7A11), tubulin (1:5000, Proteintech, 66031-1-Ig), β-actin (1:5000, Proteintech, 66009-1-Ig), Myc (1:1000, abmart, #284566), IκBα (1:2000, Cell signaling, #4814), TRAF6 (1:500, Santa Cruz, sc-8409), Ubiquitin (1:1000, Santa Cruz, sc-8017), IKK (1:1000, Cell signaling, #2682), and pS176/18-IKK (1:1000, Cell signaling, #2697) have been used.

p62 (D-3), LIMD1 (H-4), and caspase-3 (E-8) mouse monoclonal antibodies were from Santa Cruz. Mouse LIMD1(3F2C6), rabbit LIMD1, and rabbit LIMD1(N1-50) antibodies were purchased from Millipore or Sigma for different purposes. p65 (clone 2A12A7) for Amnis analysis was from Invitrogen. LMP1 (CS1-4) and TRAF6 (1H4L2) were from Dako and ABfinity, respectively. Phospho-specific antibodies and corresponding plain antibodies, K63-Ub, and horseradish peroxidase (HRP)-coupled secondary antibodies were from Cell Signaling Technologies. Mouse HA (clone HA-7), rabbit HA, and Flag (clone M2) antibodies were from Sigma. All other antibodies are from Invitrogen or eBioscience.
The human p62(−1781/+46)-Luc2 construct was provided by Terje Johansen (27 (link), 34 (link)). HA-p62 and deletion mutants (m1, m2, and m4) were from Yu-Ying He, Myc-p62(m4) was from Ying Zhao (69 (link), 70 (link)), and Flag-tagged p62 point mutants were from Ronggui Hu (51 (link)). TRAF6 and deletion mutants were from Ying Zhu (71 (link)). Additional p62 mutants were generated by site-directed mutagenesis and verified by sequencing.
Chemical inhibitors, including BAY11-7085, PD169316, and etoposide, were purchased from Sigma and EMD Millipore.