Pierce detergent compatible bradford assay

Cells treated with DMSO (the negative control) or terfenadine (1, 2, 3 μM) cells were harvested after 48 h and processed to obtain a total protein extract according to an earlier report [54 (link)]. Briefly, the parasites were collected by cooling and posterior centrifugation at 1973× g for 10 min at 4 °C. The cells were lysed with RIPA buffer (sodium chloride, 150 mM, Tris-HCL, 50 mM, Nonidet P-40 1%, sodium deoxycholate 0.5%, SDS 0.1%) supplemented with complete protease inhibitor (Roche), PMSF (Sigma-Aldrich St. Louis, MO, USA), and sodium orthovanadate (Sigma-Aldrich St. Louis, MO, USA) and were incubated on ice for 30 min. Protein concentration was determined using a Bradford assay (Pierce Detergent Compatible Bradford Assay, Thermo Fisher Scientific). Readings were made using a microplate reader at 595 nm (Multiskan SkyHigh Microplate Spectrophotometer, A51119500C, Thermo Scientific, Waltham, MA, USA). Protein extracts (25 µg) were separated under reducing conditions by electrophoresis (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, SDS-PAGE) (10%) [55 (link)]. The electrophoretic separation of the proteins was carried out at a constant voltage of 120 V for 2 h. Visualization of protein bands was carried out by incubating the gel with a Coomassie staining solution.

To measure total cellular cGAMP DN3/4 thymocytes were expanded in OP9-DL1 culture for 3 days. Cells were then harvested, washed with PBS, and lysed in 100 µl MPER buffer (ThermoFisher Scientific) for 15 min at room temperature. Lysates were cleared by centrifugation at 16,000 × g for 15 min. Total protein content for lysates was determined using Pierce Detergent-Compatible Bradford Assay (ThermoFisher Scientific) for normalization of cGAMP. cGAMP ELISAs performed using the 2′,3′-cGAMP ELISA (Cayman Chemicals) according to the manufacturer’s recommended protocol.