Ab124784

Cells were lyzed using RIPA buffer (#P0013C, Beyotime, Shanghai, China) supplemented with Protease and Phosphatase Inhibitors (#A32959, Thermo). Proteins were transferred to PVDF membranes after being separated in SDS/PAGE. The primary antibodies were incubated overnight at 4 °C, and the HRP-conjugated secondary antibodies were incubated for 1–2 h at room temperature. Final detection was performed by using ECL Plus Western Blotting Detection Reagents (#WBULS0500, Millipore). Antibodies against TAZ (#4883), β-catenin (#8480), p-ATR (#2853), p-ATM (#5883), p-BRCA1 (#9009), p-CHK1 (#2348), p-CHK2 (Thr1079, #8654) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p21 (#sc-6246), hTERT (#sc-393013), Rad51C (#sc-56214) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against TRF1 (#ab129177), TRF2 (#ab108997), POT1 (#ab124784), TIN2 (#ab197894), RAP1 (#ab175329) were obtained from Abcam (Cambridge, MA, USA). Antibodies against GAPDH (#HRP-6004), β-actin (#HRP-60008) were obtained from Proteintech Group Inc. (Wuhan, China).

The experimental binding conditions of the WEMSA were quasi-identical to the EMSA except that the total concentration of the telomeric DNA oligonucleotide was raised from 10 pM to 10 or 20 nM by supplementing the reactions with unlabeled telomeric DNA of identical sequence. Subsequent to native PAGE fractionation, the nucleic acid–protein complexes were electroblotted (20 V, 120 min, 4° C) onto a PVDF membrane under non-denaturing (0.5× TB) conditions. The PVDF membrane was soaked in 10% acetic acid for 15 min and subsequently air-dried to denature and fix the proteins to the membrane. The nucleic acid–protein complexes were firstly detected by phosphorimaging. In a second step, the proteins bound to the membrane were identified by immunoblotting. Antibody binding was visualised using WesternBright enhanced chemiluminescence HRP substrate (Advanstra) and detected by a CCD camera imager (ChemiDoc Touch, Bio-Rad) (S7A Fig). Tight specificity of the TIN2 (ab197894, Abcam), TPP1 (ab112050, Abcam) and POT1 (ab124784, Abcam) antibodies for their respective antigen and the absence of cross-reactivity with the other components of the complex was verified beforehand (S7B Fig).