Ab48614

Paraffin-embedded tissue samples were stained immunohistochemically with antibodies to ceruloplasmin (ab48614, Abcam, Cambridge, MA, USA, 1:200) according to the manufacturer's instructions. Immunohistochemical staining results were reviewed by a pathologist specializing in biliary-pancreatic disease with more than 10 years of experience. The intensity of staining was evaluated as a score of 0, 1, or 2+ for no staining, weak staining, and strong staining, respectively.

EGCG was obtained from Ebeikar Tea & Extracts Co., Ltd. (Hangzhou, China). Melatonin, quercetin (Que), luteolin, chlorogenic acid, DEDTC, 2′, 7′-dichlorofluorescin diacetate (DCFH-DA), coumarin-3-carboxylic acid (3-CCA), bathocuproinedisulfonic acid disodium salt (BCS), neocuproine, catalase, mannitol, superoxide dismutase (SOD), histidine, and methionine were purchased from Sigma (St. Louis, Missouri, USA). Plasmid pBR322 DNA was obtained from New England Biolabs (Beijing, China). ECL Plus reagent and Polyvinylidene difluoride (PVDF) membrane were products of Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The primary antibodies against γ-H2AX (sc-101696) and anti-mouse (sc-2489) secondary antibody were obtained from Santa Cruz (Dallas, TX, USA). The primary antibodies against β-actin (A5441) were purchased from Sigma. The primary antibodies against cleaved caspase 3 (9662), cleaved caspase 8 (8592), PARP (9542) and anti-rabbit (7074) secondary antibodies were products of Cell Signal Technology, Inc. (Danvers, MA, USA). The primary antibody against thrombospondin-1 (TSP-1) (ab85762) and ceruloplasmin (ab48614) were products of Abcam (Cambridge, UK). Other chemicals were of the highest grade available.

The Western blot results were verified in five patient samples (Table S10) and repeated at least twice. The human normal kidney (100mg) and ccRCC (106mg) tissues were lysed with RIPA lysis buffer containing both protease Inhibitor and phosphatase inhibitor on ice. We collected the supernatant and used a BSA Quantification Kit to determine the protein concentrations after centrifugation at 12,000 rpm for 10 minutes. Protein samples (40mg) from supernatants were separated on SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membrane was blocked for 1 hour with blocking buffer containing 5% nonfat milk. After three times washings with TBST, membranes were incubated at 4°C overnight with primary antibodies, anti-Ceruloplasmin (rabbit anti-human, 1:1000, abcam, ab48614) and anti-GAPDH (mouse anti-human/mouse/rat, 1:5000, abcam, ab8245). The membranes were washed three times with TBST and incubated with secondary antibodies at room temperature for 1 hour, and then washed three times again. Immunoreactivity was visualized by an imager (ImageQuant LAS 500; GE Healthcare). GAPDH was used for a loading control. The presented results are from at least three repetitions of Western blot. Except the primary antibody and GAPDH were Abcam, the other reagents were used the western blotting kit from BOSTER Biological Technology co. Itd (AR0040).