Immortalized WT and Sarm−/− BMDMs (A-C), immortalized WT BMDMs (D-F) or NIH3T3 cells (G,H) were stably transfected with SARM_724-Flag (S724), SARM_764-Flag (S764) or pMSCV empty vector (EV) control using a retroviral system. Cells were stimulated for 24h with 1ng/ml LPS (A), 100ng/ml LPS (D,G), 5μg/ml CLO75 (B,E) or medium as control (mock), and supernatants were assayed for CCL5 protein by ELISA. The data are mean ± SD of triplicate samples and are representative of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Sarm−/− EV (A,B) or WT EV (D,E,G) (Student's t-test). C,F,H, Cell lysates were subjected to SDS-PAGE and immunoblotted for SARM using a specific antibody to Flag, or β-actin as a loading control. The molecular weight markers are indicated to the left of the gel (in kDa).