Anti myd88 d80f5

A total of 10⁶ neutrophils were lysed and analyzed by Western blotting as previously described (26 (link)) using the following antibodies: anti-IL-1β (3ZD; National Cancer Institute Biological Resources), anti-NLRP3 (D2P5E; Cell Signaling), anti-caspase-1 (Cell Signaling), anti-NF-κB p65 (D14E12; Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1; Cell Signaling), anti-IκBα (L35A5; Cell Signaling), anti-MyD88 (D80F5; Cell Signaling), anti-TRAF6 (D21G3; Cell Signaling), anti-IKKα (Cell Signaling), anti-IKKβ (2C8; Cell Signaling), anti-phospho-IKKα/β (Ser176/180) (16A6; Cell Signaling), anti-CREB1 (48H2; Cell Signaling), anti-phospho-CREB (Ser133) (87G3; Cell Signaling), anti-C/EBPβ (Cell Signaling), anti-phospho-C/EBPβ (Thr235) (Cell Signaling), or anti-β-actin (AC-15; Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were used (BioLegend). Membranes were developed using ECL (Thermo Scientific) and detected using a Nikon camera as previously described (79 (link)). Quantification analysis of blots was performed using ImageJ, and β-actin was used as a loading control. The results for samples were expressed as a percentage of the value for the positive control (LPS or LPS+ATP group).

Western blot analysis was used to detect protein expression levels in heart tissues or cells. Briefly, heart tissues or cells were lysed in pre-cold RIPA buffer (c1053-100, APPLYGEN, China) with a 0.5% protease inhibitor (p1625, APPLYGEN, China) and 1% phosphatase inhibitor (p1260-1, APPLYGEN, China). The protein concentration of each sample was measured using a BCA kit (P1511-1, APPLYGEN, China). Equal quantities of the proteins from each group were loaded on 10% PAGE gel fast preparation kit (PG112, Epizyme, China), electrophoresed, and transferred onto PVDF membranes (p2120-6, APPLYGEN, China). The membranes were incubated with primary and secondary antibodies and then treated with ECL (SQ201, Epizyme, China). The following antibodies were used: anti-TLR4 (19811-1-AP, Proteintech, United States), anti-MyD88 (D80F5, Cell Signaling Technology, Germany), Phospho-IκBα (Ser32) (14D4, Cell Signaling Technology, Germany), anti-IKBα (ab32518, Abcam, United States), Phospho-NF-κB p65 (Ser S536) (93H1, Cell Signaling Technology, Germany), anti-NF-κB p65 (D14E12, Cell Signaling Technology, Germany), and anti-GAPDH (ab8246, Abcam, United States), anti-mouse IgG H&L (AB0102; Abways, China) and anti-rabbit IgG H&L (AB0141; Abways, China). Finally, Image Lab software was used for band gray analysis.