Ab182453

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).

The Western blots were performed as described in the previous study [1 (link),49 (link)]. The liver and gut tissue homogenates were solubilized in sodium dodecyl sulfate (SDS) sample buffer. Samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Appropriate primary antibodies were used for incubating overnight. Antibodies against mitochondrial respiratory chain complex I (NADPH-diaphorase) (SC-20493), II (complexII) (sc65239), complex III (cytochrome reductase) (SC-69064), β-actin (SC-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against CYP2E1 (ab28146), Cry 1 (ab54649), α-tubulin (ab7291), p-NF-kB (ab76302), NF-kB (ab76311), p38 (ab182453), p-p38 (ab178867), JNK (ab199380), p-JNK (ab307802) were purchased from Abcam (Abcam, Cambridge, MA, USA). The dilution concentration of the primary antibody is 1:1000. The immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) reagent after incubating appropriate secondary antibodies (dilution concentration is 1:5000). Quantification of Western blot results using band densitometry analysis was performed with Quantity One software 4.6.2.