Anti traf1

Subcellular fractionation was performed with a Nuclear and Cytoplasmic Protein Extraction Kit (Sangon, Shanghai, China), in accordance with the manufacturer’s instructions. Total protein separation and western blotting were performed as described previously [30 (link)]. The following antibodies were used: anti-p-IκBα (Cell Signaling Technology, USA), anti-p-IKKβ (Abcam, Cambridge, MA, USA), anti-β-actin, anti-Lamin B1, anti-TRAF1 and anti-NF-κB p65 (Proteintech, Wuhan, China). All experiments were performed in triplicate.

IHC was performed following a previously described method [30 (link)]. The following antibodies were used: anti-CD34, anti-VEGF, anti-DNMT1, anti-TRAF1, and anti-NF-κB p65 (Proteintech, Wuhan, China). Staining intensity was scored manually by two independent experienced pathologists as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The percentage of positive cells was also assessed according to four scores: 1 (0-10%), 2 (11-50%), 3 (51-80%), and 4 (81-100%). The final IHC score was calculated by multiplying the intensity score by the percentage of positive cells. CD34 antibody was used to stain vascular endothelial cells, and then the microvessel density (MVD) was calculated. The field of maximal CD34 expression was found in tumor tissues. Within this field, the area of maximal angiogenesis was selected, and microvessels were counted at 200× magnification. Low MVD was indicated by scores from 0 to 3. High MVD was indicated by scores ≥4 [31 (link)].

Liver tissue was lysed on ice with RIPA lysis buffer, and protease inhibitors, phosphatase inhibitors and 1mM PMSF (Beyotime, China) were added. The extracts were centrifuged at 12,000 rpm (8000 × g) and 4°C for 15 min to remove insoluble material, and the supernatants were used for protein quantification, performed by the BCA method. Equal amounts of proteins (30–90 μg) were separated on 8%–12% SDS‐PAGE gels, and then transferred onto PVDF membranes (Beyotime, China). Membranes were subsequently blocked with 5% skimmed milk. The membranes were incubated with following primary antibodies: anti‐TRAF1, anti‐CFLAR, anti‐DKK3, anti‐CREG anti‐Card6 and anti‐ASK1 from Proteintech (USA); anti‐phospho‐ASK1 (Thr845), anti‐SAPK/JNK, anti‐phospho‐SAPK/JNK (Thr183/Tyr185), anti‐IRS1, anti‐phospho‐IRS1 (Ser307) and anti‐Akt from Cell Signaling Technology (USA), anti‐phospho‐Akt (Ser473) from Abcam (USA); anti‐GAPDH from Sangon Biotech (China). After washing with 0.1% Tween 20 in TBS, the membranes were incubated with HRP‐conjugated secondary antibody (Sangon Biotech) and bound antibody was detected by BeyoECL Star (Beyotime, China). Bands were quantified and analysed by Image Pro Plus software.