Peroxidase conjugated affinipure goat anti mouse igg h l

Total protein was extracted by lysing cells in RIPA buffer containing protease inhibitors. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in TBST, membranes were incubated with the primary antibody. Antibodies against ZEB1, cleaved caspased-3, E-cadherin, N-cadherin, Vimentin, Snail and Slug purchased from Cell Signaling Technology in USA were diluted at 1:1000. Bcl-2, Bax, PCNA, UBQLN1, MMP2, MMP9 and β-actin purchased from Proteintech Group in USA were diluted at 1:1000 and Peroxidase-conjugated Affinipure goat anti-mouse IgG (H+L) or anti-rabbit IgG (H+L) were diluted at 1:4000 as the secondary antibody.

Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐Rabbit IgG HRP‐linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).

Daphnetin (purity>98%, catalog number:110900) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Daphnetin was diluted in culture medium with 0.1% dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA) and then sterile-filtered before used. All controls and samples contained the same concentrations of DMSO.
Concanavalin A (ConA) and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The mAbs against mouse CD3, CD4, CD8, IFN-γ, IL-2, IL-4 and IL-6 ELISA kits were purchased from Biolegend (California, USA). RPMI-1640 medium was purchased from HyClone (Logan, UT), and fetal bovine serum (FBS) was obtained from Invitrogen-Gibco (Grand Island, NY). Primary antibodies used for Western analysis Calmodulin, Calcineurin (CaN), Ca²⁺/Calmodulin-dependent kinase II (CaMKII), P-CaMKII, NF-κB, which were purchased from Cell Signaling (beverly, MA), NFAT2 antibodies was purchased from Abcam (Cambridge, MA), β-actin was obtained from Tianjin Sungene Biotech Co., Ltd. Goat anti-mouse IgG, Goat anti-mouse FITC, Peroxidase-conjugated AffiniPure Goat anti-mouse IgG (H + L) were purchased from Protein Tech Group, 2, 4-Dinitrofluorobenzene (DNFB) and cyclophosphamide (CTX) were obtained from Sigma-Aldrich (St. Louis, MO).