After the LPS and oridonin treatment for 24 h, cells from each group were collected and lysed on ice for 20 min by adding RIPA lysate (Solarbio). Next, the lysed cells were centrifuged at 10,000 rpm, 4 °C for 15 min to collect proteins and the protein concentrations were detected through a BCA kit (Solarbio). There was 30 μg of protein taken and denatured at 95 °C for 15 min with addition of the protein loading buffer. Later, the protein bands were separated by SDS PAGE electrophoresis, and then the protein was transferred to PVDF membranes and blocked in 5% skimmed milk blocking solution for 1–3 h. Subsequently, the membranes were washed three times in TBST buffer and then incubated overnight at 4 °C on a shaker with the addition of diluted primary antibodies (p65, ab16502, Abcam; p-p65, ab76302, Abcam; NLRP3, ab263899, Abcam; Caspase-1, ab138483, Abcam; GRP78, ab21685, Abcam; CHOP, #5554, CST; ATF4, ab184909, Abcam; ATF6, ab122897, Abcam; β-actin, ab6276, Abcam, UK). Afterwards, the membranes were washed with TBST twice, and diluted secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., China) was added for 1-h incubation at ambient temperature. Again, the membranes were washed with TBST three times, and ECL hypersensitive luminescence solution (Solarbio) was added dropwise to develop the protein bands in a gel imager for photography. The protein bands were measured in grayscale employing Image J software and finally, the relative expression level of the target protein was analyzed with β-actin as an internal control.
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