Human gapdh

Whole cell lysates were harvested with cell lysis buffer according to the manufacturer’s instructions. Western blotting analyses were performed with the standard protocol using antibodies against human GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3 (Novus, Littleton, CO, USA) and P62 (Cell Signaling Technology, Beverly, MA, USA).

Western blot was performed as described previously [9 (link)]. Briefly, total protein of DPCs at P1 to P7 was measured by Bio-Rad Coomassie Blue protein assay (Bio-Rad Laboratories, Richmond, CA, USA), whereas only the representative results of P3 and P7 were presented in Results. Twenty micrograms of protein was diluted by 10% bromophenol blue and boiled before being separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked in 5% low-fat milk at room temperature for 1 h, rinsed, and incubated with monoclonal antibodies mouse against human XPC (1 : 500 dilution, Abcam, MA, USA), Oct-4 (1 : 100, Chemicon, MA, USA), Sox2 (1 : 50, R&D System, MN, USA), c-Myc (1 : 50, Santa Cruz, CA, USA), or human GAPDH (1 : 1000 dilution, Santa Cruz, CA, USA) overnight at 4°C. After washing, the membrane was incubated with the HRP-conjugated secondary antibody (1 : 5000 dilution, Jackson, USA) at room temperature for 1 h. Immunoreactive proteins were then visualized by incubating membranes with electrogenerated chemiluminescence plus detection agents (GE Healthcare, USA).

For SDS-PAGE cell or tissue lysis was performed using RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% (v/v) Nonidet P40, 0.1% (w/v) SDS, 10 μM MG132, 5 mM NEM, Complete^® protease inhibitor cocktail (Roche)).
For native-PAGE, cells were lysed with TSDG buffer (10 mM Tris-HCl pH 7.0, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl₂, 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol) followed by 4 cycles of snap freezing and thawing.
Immunoblot analysis was performed according to standard procedures [34 (link)]. Primary antibodies: murine PA28α (K232/2, lab stock), murine PA28β (K231/2, lab stock), human PA28α (Cell Signaling), human PA28β (Cell Signaling), α4 (K378/1, lab stock), LMP7 (K63, lab stock), Rpt6 (Enzo Lifesciences), VP1 (DAKO), Ubiquitin (DAKO), DNP (Sigma), actin (Millipore), human GAPDH (Santa Cruz), mouse GAPGH (GeneTex). The bound primary antibodies were detected using either (i) IRDye800CW labelled goat anti-mouse/anti-rabbit secondary antibodies in conjunction with an Odyssey CLx infrared imaging system (Li-Cor Biosciences) or (ii) horseradish peroxidase (HRP)-labelled goat anti-mouse/anti-rabbit secondary antibodies (Santa Cruz) using an enhanced chemiluminescence light (ECL) detection kit (GE Healthcare) and a PEQLAB Fusion FX Imaging System.