Abx120550

Anti-hTERT mouse monoclonal antibodies (mAbs, clones 10E9-2 and 2E4-2) were generated and described the specificity as reported previously^{14 (link)}. Phosphospecific rabbit polyclonal antibodies (pAbs) and mouse mAb against phosphorylated threonine 249 of hTERT (anti-249T-P and TpMab-1, respectively) were generated in this study (see below). Anti-hTERT mouse mAb (1:100, clone 2E4-2), anti-hTERT rabbit mAb (1:1000, Abcam, ab32020), anti-249T-P pAbs (1:1000), TpMab-1 mAb (1:50), anti-phospho-histone H3 (Ser10) rabbit pAbs (1:2000, Merck, 06-570), anti-cdc2 [CDK1] (POH1) mouse mAb (1:1000, Cell Signaling Technology, 9116), anti-β-actin (AC-15) mouse mAb (1:20000, Sigma-Aldrich, A5441), anti-FOXO4 rabbit mAb (1:4000, Abcam, ab128908), anti-GAPDH mouse mAb (1:1000, MBL Co., Ltd., M171-3), and anti-SMG6 rabbit pAbs^{64 (link)} (1:1000) were used for immunoblotting (IB). Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP). Anti-CDK1 rabbit pAbs (1:250, Merck, HPA003387), anti-249T-P pAbs (1:250) and TpMab-1 mAb (1:250) were used for immunohistochemistry.

Western blot analysis was performed as described previously (16 (link)). Briefly, samples were separated on SDS–polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with corresponding antibodies. Immunoreactive bands were visualized using the ChemiDoc™ Imaging System (Bio-Rad, USA) with a Super Signal™ West Pico PLUS Chemiluminescent Substrate Kit (Thermo). The primary antibodies included anti-LDHB (ab53292, Abcam, USA), anti-LDHA (66287-1-Ig, Proteintech, China), anti-Flag (A8592, Sigma, USA), anti-Myc (M047-7, MBL, USA), β-actin (sc-47778HRP, Santa Cruz, USA), anti-PKM2 (15822-1-AP, Proteintech, China), anti-HK2 (66974-1-Ig, Proteintech, China), anti-PGK1 (17811-1-AP, Proteintech, China), anti-ENO1 (11204-1-AP, Proteintech, China), anti-TERT for WB (ab32020, Abcam, USA), anti-TERT for IP (abx120550, Abbexa, United Kingdom).