Ab75782

Total protein from cell lines was extracted using Radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail. Protein concentrations were determined by a BCA protein assay kit (P0009; Beyotime, Shanghai, China). Equal amounts of protein were separated via denaturing Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes and blocked, and then incubated overnight at 4°C with primary antibodies directed against HMGB3 (ab75782; Abcam), CD36 (18836-1-AP; Proteintech, Manchester, UK), E-cadherin (14472 S, CST), Snail (3879 S, cell signaling technology [CST]), vimentin (SK2482712C, Invitrogen), and β-actin. Corresponding horseradish peroxidase-conjugated secondary antibody was subsequently added for 2 hours at room temperature, and protein bands were visualized by exposure to film. The relative protein levels were calculated based on β-actin as the loading control.

Antibodies against glutathione-S-transferase (GST, 10000-0-AP), poly (ADP-ribose) polymerase 1 (PARP1, 13371-1-AP), Flag (20543-1-AP), MYC-tag (16286-1-AP), GFP (66002-1-Ig), Ki-67 (27309-1-AP), histone-H3 (17168-1-AP), topoisomerase 1 (TOP1, 20705-1-AP), and caspase 3 (19677-1-AP) were purchased from Proteintech (Wuhan, China); PAR (AM80), β-actin (A5441), and methyl methanesulfonate (MMS, M4016) were obtained from Sigma–Aldrich (St. Louis, MO, USA); mouse IgG (A7028) and rabbit IgG (A7016) were purchased from Beyotime (Shanghai, China). Antibodies for γH2AX (ab22551), H2AX (ab229914), HMGB3 (ab75782), and Rad51 (ab63801) were obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG Alexa Fluor-488 (A-11008) and Goat anti-mouse IgG Alexa Fluor-594 (A-11030) for immunofluorescence staining were obtained from Invitrogen (Waltham, MA, USA). Olaparib (AZD2281) was purchased from Selleck Chemicals (Houston, TX, USA).

The total protein of lung tissues and BEAS-2B cells were extracted and then quantified with bicinchoninic acid assay (Pierce, Rockford, USA). Then, it was resolved on 10% polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Afterward, the PVDF membranes were blocked with 5% skim milk at RT for 1 h and incubated with the primary Anti-NF-κB p65 (phospho S276) antibody (1:1000, ab222494, Abcam, USA), Anti-NF-κB p65 antibody (1:1000, ab16502, Abcam, USA), Anti-MAPK p38 (phospho T180) antibody(1:1000, ab178867, Abcam, USA), Anti-MAPK p38 antibody(1:1000, ab31828, Abcam, USA), Anti-iNOS antibody(1:1000, ab178945, Abcam, USA), Anti-COX2 antibody (1:1000, ab179800, Abcam, USA), Anti-HMG4 antibody (1:1000, ab75782, Abcam, USA), Anti-β-actin antibody (1:1000, ab8224, Abcam, USA) and Anti-GAPDH antibody (1:1000, ab181602, Abcam, USA) overnight at 4 °C. Subsequently, the membranes were washed twice with TBST, and then incubated with the fluorescein-labeled secondary Goat Anti-Rabbit IgG (1:3000, ab150077, Abcam, USA) at RT for 1 h. After that, the membranes were rinsed 3 times. The protein bands were examined by the ECL WB kit (Amersham Biosciences, UK). The primary images of the blots were shown in the Additional file 1: Fig. S1, Additional file 2: Fig. S2 and Additional file 3: Fig. S3.

After being fixed and decalcified, the blocks were processed into routine slices and then underwent dewaxing with xylene and hydration with gradient alcohol. The slices were applied with 3% H2O2, followed by 15-min resting to blockade endogenous peroxidase activities remaining in tissues, and then were washed with PBS triply. The ccRCC slices were applied with the citrate solution (10 mM) and heated in microwave oven for 20 min for antigen retrieval. The ccRCC slices were subsequently applied with 5% serum of goat for 10 min, for the sake of blocking the nonspecific binding. After applying with the primary antibodies of CD8 (1/200, ab4055, Abcam, UK), CD45RO (5 μg/ml, ab23, Abcam), HMGB3 (1/100, ab75782, Abcam), IKBKE (10 μg/ml, ab7891, Abcam), PLTP (1/50, ab189776, Abcam), PTGES (10 μg/ml, ab62050, Abcam) and CD36(1:100 ab133625, Abcam), the slices were preserved at 37°C for 0.5 hour and rinsed with PBS. After applying with the secondary antibody (18653, Cell Signaling Technologies, USA) labelled horseradish peroxidase, the slices were preserved at 37°C for 0.5 hour and rinsed with PBS once again, stained utilizing the diaminobenzidine (DAB) (91-95-2, Sigma-Aldrich Chemical Company, USA) for 10 min, rinsed with running purified water, stained with hematoxylin, added with neutral resins and eventually observed utilizing the microscope and the pictures were taken.

UWB1.289 (1 × 10⁷) cell protein lysates were prepared using Western and IP Lysis Buffer, followed by centrifugation at 4 °C for 15 min. The cell supernatant was immunoprecipitated (IPed) using anti-IgG or anti-HMGB3 (ab75782, Abcam) antibody. The IPed proteins were resuspended with 2 × SDS loading buffer and subjected to SDS-PAGE assay. Gels were stained with coomassie brilliant blue, and pieces were removed for MS analysis. Liquid chromatography-tandem MS (LC-MS/MS) analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) by Applied Protein Technology (Shanghai, China).