Rabbit anti human pcna

Sections were incubated with antibody of COX IV (1: 200, Abcam) or rabbit anti-human PCNA (1: 2000, Cell Signaling Technology). The visualization signal was developed with the Invitrogen Histostain Plus kit. The intensity and extent of COX IV immunostaining were evaluated for all samples under double-blinded conditions. In brief, the percentage of positive staining was scored as 0 (0–9%), 1 (10–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%), and the intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (dark staining). The total score was calculated as the product of intensity and extent, ranging from 0 to 12.

Immunoblot was performed as previously described.20 (link) A total of 50 µg of protein was transferred onto PVDF membranes by electrophoretic transfer following electrophoretic separation by 12% SDS-PAGE. The antibodies used were rabbit-anti human Pim1 (1:1,000; Cell Signaling), rabbit-anti human PCNA (1:1,000; Cell Signaling), mouse-anti LMP1 (1:500; Abcam, Cambridge, UK) and rabbit-anti human β-actin (1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were incubated overnight at 4°C, washed with tris-buffered saline-Tween (TBST), and incubated with HRP-labeled IgG (1:3,000; ProteinTech, Wuhan, China) for 2 hours at room temperature. After ECL luminescence, the target protein bands were scanned with gel imaging system (Tanon, Shanghai, China).