Gene expression and protein analysis was performed as described by Takahashi et al. [8 (link)]. For the quantification of mRNA, TaqMan real-time polymerase chain reaction (PCR) amplification reactions were performed using an Applied Biosystems 7500 Fast Real-Time System (Thermo Fisher Scientific Inc., Waltham, MA). All primers and the TaqMan Universal PCR Master Mix were obtained from Thermo Fisher Scientific. Specific primers for the mouse genes Gapdh (Mm99999915_g1), interleukin- (IL-) 6 (Mm00446190_m1), TNFα (Mm00443258_m1), and IL-1β (Mm00434228_m1) were used. Gene expression levels were normalized to the Gapdh expression level.
To measure the protein expression, the amounts of IL-6 and IL-1β in RAW264 cells were assayed in the supernatant of the cell culture medium using the Bio-Plex Pro™ Mouse Cytokine Assay kit (BIO-RAD, USA) with a MAGPIX xPONENT 4.2 system (Merck Millipore Co, USA).
To measure the protein expression, the amounts of IL-6 and IL-1β in RAW264 cells were assayed in the supernatant of the cell culture medium using the Bio-Plex Pro™ Mouse Cytokine Assay kit (BIO-RAD, USA) with a MAGPIX xPONENT 4.2 system (Merck Millipore Co, USA).
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