Epr19759

A tissue microarray (TMA) containing 756 primary ccRCC and corresponding normal tissue samples was used as described before [28] (link). For immunohistochemical staining of tissue, microarray slides or formalin fixed and paraffin-embedded tumor tissue antibodies directed against CD3 (IR503, Dako, Glostrup, Denmark), CD8 (C8/144B, Dako), CD20 (IR604, Dako), CD56 (123C3, Dako), CD68 (PG-M1, Dako), CD138 (MI15, Dako), FoxP3 (236A/E7, Abcam, Cambridge, United Kingdom), myeloperoxidase (MPO) (IR511, Dako), PD-1 (NAT105, Abcam), and PD-L1 (EPR19759, Abcam) were used. All slides were stained with automatized immunostainers (autostainer plus, Dako).

Anti‐PD‐L1 antibody against the extracellular domain of human PD‐L1 was purchased from Abcam (EPR19759; Abcam, Cambridge, UK). Anti‐GAPDH antibody and peroxidase‐conjugated secondary antibody were the products of ZhongShan Golden Bridge Biotech Co (Beijing, China). The human CRC cell lines SW620, SW480, and HCT8 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).

Whole-cell lysates were prepared as previously described.6 7 (link) Equal amounts of protein (10–50 µg) were resolved by 12% SDS-PAGE. After electrophoresis, separated proteins were transferred onto the nitrocellulose membrane. The membrane was blocked in 5% non-fat milk, followed by overnight incubation with primary antibodies. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the positive immune reactive signal was detected by enhanced chemiluminescence (ECL; Fude Biotech, Hangzhou, China). Antibodies specific for TSHβ (D-6, 1:500), TSHR (C-10, 1:500), and β-actin (sc-47778, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies specific for PD-L1 (EPR19759, 1:250) and total p65 (E379, 1:1000) were obtained from Abcam (Cambridge, UK). Antibodies specific for total c-JUN (9165, 1:1000), phosphorylated c-JUN (3270, 1:1000), total AKT (4685, 1:1000), phosphorylated AKT (4060, 1:1000), phosphorylated ERK (4370, 1:1000), phosphorylated JNK (4668, 1:1000), phosphorylated p38 (4511, 1:1000) and phosphorylated p65 (3033, 1:1000) were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Antibodies specific for TSHα (25014–1-AP, 1:1000), total ERK (16443–1-AP, 1:1000), total JNK (24164–1-AP, 1:1000), total p38 (14064–1-AP, 1:1000) and HIF1α (66730–1-Ig, 1:1000) were purchased from Proteintech.