Inos and cox 2

Approximately 3 × 10 5 cells were seeded in six-well plates. The cells were cultured and treated as aforementioned. The RAW 264.7 cells were washed twice with phosphate buffered saline (PBS) and then lysed with CHAPS (Amresco) plus protease inhibitors. Aliquots containing 30 μg proteins from each sample were run on a 10% sodium dodecylsulphate polyacrylamide gel. After running the gel, the proteins were transferred to a nitrocellulose membrane (Sartorius, Germany) and then, the blots were blocked with Tris buffered salinea plus 5% nonfat dry milk and 0.05% Tween-20 for 1 hr. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (iNOS and COX-2; Cell Signaling) and α-tubulin (Cell Signaling) at 1:500 and 1:1,000, respectively. Later, the blots were incubated with secondary antibodies containing peroxidase-conjugated (1:2,000) for 1 hr at room temperature. The blots were analyzed by chemiluminescence, and digital images were caught using a luminescent image analyzer, Carestream Gel Logic 2200 PRO Imaging System. The total expression was evaluated in Image Studio Lite software (LI-COR) and, α-tubulin was used for normalization of quantitative densitometry values (de Mesquita et al., 2017; (link)Tripathi et al., 2018) (link).