Phosphorylation-Specific Antibody Development

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.