P38 mapk

Cells were harvested, and equal amounts of protein were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gels for 2 h at 100 V and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was blocked with 5% skim milk for 1 h at room temperature. After washing three times with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST), the membranes were incubated with appropriately diluted Phospho-p44/42 MAPK, p44/42 MAPK, Phospho-JNK/SAPK, JNK/SAPK, Phospho-p38 MAPK, and p38 MAPK (Beyotime, Shanghai, China) antibodies at 4 °C overnight. Then, the membranes were washed as before and incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit) for 1 h at room temperature. After that, these membranes were washed thoroughly, to eliminate the unspecific antibody. At last, proteins were detected using enhanced chemiluminescence (ECL) blotting reagents according to the manufacturer’s instruction.

Neochlorogenic acid (nCGA, purity >98%) was isolated from mulberry leaf (Morus alba L.), and the chemical structure was identified in our laboratory [45 (link)] (Figure 7). A 10 mM stock solution of nCGA was prepared in dimethyl sulfoxide (DMSO).
Lipopolysaccharide (Escherichia coli 055:B5), MTT and dimethylsulfoximine (DMSO) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and RPMI 1640 medium were obtained from HyClone (Logan, Utah, U.S.). Human ELISA kits were purchased form Biolegend (San Diego, CA, USA). Compound C (AMPK inhibitor) was purchased from Sigma (St. Louis, MO, USA). Primary antibodies against phospho-ERK1/2, ERK1/2, phospho-p38 MAPK, p38 MAPK, JNK, phospho-JNK, p65/p-p65, IκBα, and β-actin were purchased from Beyotime Institute of Biotechnology (Beijing, China), primary antibodies against TNF-α, IL-6, iNOS, and COX2 were obtained from Biolegend (San Diego, CA, USA). Second antibodies and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) were obtained from Beyotime Institute of Biotechnology (Beijing, China). All other chemicals were of reagent grade.

Immunoblotting analysis was performed as described.⁹ 5% BSA and non‐fat milk were used as blocking reagent. The corresponding antibodies mainly included ZCCHC14 (ab‐150591, Abcam, USA), P42/44 (AM076, Beyotime, China), p‐P42/44 (AM071, Beyotime, China), cyclinD1 (AC853, Beyotime, China), MMP7 (D4H5; 3801, Cell Signaling, USA), MMP9 (D6O3H; 13 667, Cell Signaling, USA), GADPH (ab8245, Abcam, USA), JNK (AJ518; Beyotime, China), P‐JNK (AJ516, Beyotime, China), P38MAPK (AM065, Beyotime, China) and p‐P38MAPK (AM063, Beyotime, China),