Western blotting was performed as previously described [31 (link)]. Proteins were extracted from pMECs using the radio immunoprecipitation assay (RIPA) lysis buffer (#P0013B, Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (#P0009, Beyotime, Shanghai, China). Proteins (10–20 μg/sample) were separated by SDS-PAGE (Invitrogen Inc.), transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA.), and then hybridized with specific antibodies.
Primary antibodies for TLR-4 (1:500, ab13556), p65 (1:1000, ab16502), phospho-p65 (1:1000, ab183559)), β-actin (1:1000, ab5694), p38 (1:1000, ab182453), phospho-p38 (1:1000, ab207483), ERK (1:1000, ab32537), phospho-ERK (1:1000, ab207470), JNK (1:1000, ab126424), and phospho-JNK (1:1000, ab279842) were from the Abcam Company Ltd. (Cambridge, MA, USA). Primary antibodies (dilution, cat. no. follow in parentheses) for IκBα (1:1000, #9242), and phospho-IκBα (1:1000, #2859) were from the Cell Signaling Technology (Woburn, MA, USA).
Primary antibodies for TLR-4 (1:500, ab13556), p65 (1:1000, ab16502), phospho-p65 (1:1000, ab183559)), β-actin (1:1000, ab5694), p38 (1:1000, ab182453), phospho-p38 (1:1000, ab207483), ERK (1:1000, ab32537), phospho-ERK (1:1000, ab207470), JNK (1:1000, ab126424), and phospho-JNK (1:1000, ab279842) were from the Abcam Company Ltd. (Cambridge, MA, USA). Primary antibodies (dilution, cat. no. follow in parentheses) for IκBα (1:1000, #9242), and phospho-IκBα (1:1000, #2859) were from the Cell Signaling Technology (Woburn, MA, USA).
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