T1038

Most of inhibitors used were purchased from TargetMol, including T3708 (BP-1-102), T6309 (AZ960), T2814 (cryptotanshinone), T4234 (HJC0152 hydrochloride), T4216 (STAT5 inhibitor), T1038 (fludarabine), T1849 (momelotinib), T6122 (CEP-33779), T3620 (solcitinib), T3998 (itacitinib), and T4657 (WHI-P97). Only ochromycinone (STA-21) were purchased from Selleck. All inhibitors were dissolved in DMSO to a storage concentration of 10 mM and stored at −80 °C. It is important to avoid repeated freezing and thawing for inhibitor activity. Embryos from the same parent were divided into control and treatment groups at 6 hpf. Every treatment group was raised in 1X Holt buffer with all kinds of inhibitors and control groups was raised in in 1X Holt buffer. Embryos were observed and collected for the following experiments at 3 dpf. Each inhibitor treatment has at least three biological replications and three technical replications.

Purified Ccr2‐KO splenic B cells (2 × 10⁶) were incubated with rapamycin (20 nM, HY‐10219, MedChem Express), XMU‐MP‐1 (3 μM, T4212, TargetMol), fludaradine (5 μg, T1038, TargetMol) or R406 (5 μM, T6174, TargetMol) at 37°C for 2 h (R406 for 4 h) before being activated with sAg. Then western blotting was performed on WT B, Ccr2‐KO B and Ccr2‐KO plus inhibitor B cells.