Mouse anti o glcnac transferase c 10

For pluripotency marker analysis, mESCs were grown on microscope slides precoated with fibronectin, containing MEF feeders. mESCs were fixed with 4% PFA for 20 min, washed in 1% BSA, and incubated overnight with primary antibody at 4 °C. The primary antibody used in this study include mouse anti-phospho-Histone H2A.X (Ser139) (Millpore, Cat# 05–636, 1:100 dilution); mouse anti-O-linked N-acetylglucosamine (O-GlcNAc) (HGAC85) (Thermo Fisher Scientific, Cat# MA1–076, 1:50 dilution); rabbit phospho-ATM/ ATR Substrate Motif [(pS/pT) QG] (Cell signaling, Cat# 6966S, 1:100 dilution); mouse anti-O-GlcNAc transferase (C-10) (Santa Cruz Biotechnology, Cat# sc-376253, 1:100 dilution); rabbit anti-SOX2 (Cell signaling, Cat# 9656S, StemLight Pluripotency Antibody Kit, 1:50 dilution); and rabbit anti-TRA-1–60(S) (Cell signaling, Cat# 9656S, 1:50 dilution). The samples were then incubated for 2 h with secondary antibodies at 25 °C, washed in PBST, mounted with Vectashield and analyzed using a Zeiss LSM 700 system.

MEFs were grown on microscope slides precoated with fibronectin. MEFs were fixed with 4% PFA for 20 min, washed in 1% BSA, and incubated overnight with primary antibody at 4 C. The primary antibody used in this study include mouse anti-phospho-Histone H2A.X (Ser139) (Millpore, Cat# 05–636, 1:100 dilution); mouse anti-O-linked N-acetylglucosamine (O-GlcNAc) (HGAC85) (Thermo Fisher Scientific, Cat# MA1–076, 1:50 dilution); mouse anti-O-GlcNAc transferase (C-10) (Santa Cruz Biotechnology, Cat# sc376253, 1:100 dilution); rabbit anti-Ki-67 (abcam, Cat# ab15580, 1:100 dilution); and mouse anti-Ki-67 (SP6) (Thermo Fisher Scientific, Cat# MA5–14520, 1:50 dilution). The samples were then incubated for 2 h with secondary antibodies at 25 °C, washed in PBST, mounted with vectashield and analyzed using a Zeiss LSM 700 system.