P p38 mapk

A portion of LV was homogenized in ice‐cold RIPA buffer and centrifuged at 12,000 g for 15 min at 4°C. The protein concentration was evaluated by the Bradford method using bovine serum albumin as a standard. Sixty micrograms of supernatant proteins were resolved on SDS‐PAGE and transferred to PVDF membrane (2 h). The transference was confirmed by Ponceau S staining. Membranes were blocked with 5% nonfat milk and probed overnight at 4°C with antibodies against P‐p38MAPK (1:1000, Santa Cruz Biotechnology), p38MAPK (1:1000, Santa Cruz Biotechnology), P‐PKCε (1:1000, Santa Cruz Biotechnology), PKCε (1:1000, Santa Cruz Biotechnology), calcineurin Aβ (1:1000, Santa Cruz Biotechnology), P‐HSP27 (1:500, Santa Cruz Biotechnology), PSer637 Drp1 (1:1000, Cell Signaling), and Drp1 (1:1000, Santa Cruz Biotechnology). Membranes were washed four times prior to addition of anti‐mouse (1:5000, Lobov) or anti‐rabbit (1:5000, Sigma‐Aldrich) secondary antibody and protein bands were analyzed by a chemiluminescent system. GAPDH or total (phosphorylated plus non‐phosphorylated forms) protein expression was used as housekeeping protein.

To prepare liver tissue samples for Western blotting, equal amounts of protein (30 μg) were extracted and resolved on 7–12% SDS-PAGE gels. The separated proteins were then transferred to PVDF membranes (Millipore) and incubated overnight at 4 °C with primary antibodies specific to the target proteins. The following day, the membranes were incubated with anti-mouse, anti-goat, or anti-rabbit secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Primary antibodies used were COX-2 (sc-376861), iNOS (sc-7271), NF-κB p65 (sc-8008), p-ERK (sc-7383), p-p38 MAPK (sc-7973), p38 MAPK (sc-7972), p-Akt (sc-7985-r), Akt (sc-8312), PI3-kinase p110β (sc-602), Nrf2 (sc-722), HO-1 (sc-136961), AMPK (sc-25792), Histone H1 (sc-393358), and β-actin (Sc-47778), all purchased from Santa Cruz Biotechnology. Other primary antibodies used included ERK (#9102), p-JNK (#9255), JNK (#9252), and p-AMPK (#2535) from Cell Signaling Technology, and GAPDH (GTX100118) from Gene Tex. The blots were developed using an ECL detection kit (Advansta, CA, USA), and a quantitative analysis of protein levels was performed using ImageJ 1.53e software (NIH, Bethesda, MD, USA).

The transfected HUVECs were lysed with cell lysis buffer (Thermo Fisher Scientific) for total protein extraction. The concentration of protein was measured by a Bradford Protein Assay kit (Sangon Biotech, China). After vortexing for 5 min, equal amounts of protein from each sample were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked with 5% non-fat milk and probed with antibodies against SIRT6, JNK, p-JNK, p38 MAPK, or p-p38 MAPK (1:1000, Santa Cruz Bio-Technology, USA) overnight at 4°C. Then, the membranes were incubated with the secondary antibody (1:5000, Santa Cruz Bio-technology) with horseradish peroxidase conjugated antibody for 2 h at room temperature. Finally, signal intensity of complexes was visualized with an enhanced chemiluminescence detection kit (Beyotime).

The differential protein expression of Bax, Bcl-2, cleaved caspase-3, ACHE, IL-1β, TNF-α, p-P38MAPK, and p-JNK in the various groups was determined by Western blotting. Cells were lysed in 100 μl RIPA lysis buffer and PMSF mixture (Beyotime, Shanghai, China) at 4 °C for 15 min. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to determine protein concentrations. An equal amount of protein samples was separated by 4–12% SDS-PAGE and then transferred to 0.45 μm PVDF membranes (PMembrane, Beijing, China). After being blocked by 5% non-fat milk for 2 h, membranes were incubated with corresponding primary antibodies to ACHE (Servicebio, Wuhan, China), Bax (Abcam, Waltham, MA, USA), Bcl-2 (Bioss, Beijing, China), Cleaved caspase-3 (CST, San Antonio, TX, USA), IL-1β (Abcam, Waltham, MA, USA), TNF-α (Bioss, Beijing, China), p-P38MAPK (Santa Cruz, CA, USA) and p-JNK (CST, San Antonio, TX, USA) at 4 °C overnight. After that, we utilized the HRP-conjugated secondary antibodies to incubate the PVDF membranes at room temperature, and the imaging system (Analytik Jena, Jena, Germany) and enhanced chemiluminescence detection kit (Biosharp, Hefei, China) to detect the immunoblots.

All the experiments were performed using SVG astrocytes. The cells were cultured in Dulbecco’ modified Eagle’s Medium (DMEM) that was supplemented with 10% fetal bovine serum and 1% non-essential amino acids, 1% sodium bicarbonate, 1% L-glutamine and 0.1% gentamicin. The cells in culture were maintained at 37 °C with 5% CO₂. HIV1-gp120 (pSyngp120 JR-FL) plasmid used for transfection was originally constructed by Drs. Park and Seed (Department of Molecular Biology, and Melvin and Barbara Nessel Gene Therapy Center, Massachusetts General Hospital, Boston, MA, USA). Recombinant HIV-1 IIIB gp120 was obtained from NIH AIDS reagents program. Resveratrol was purchased from Sigma–Aldrich (St. Louis, MO). Resveratrol analog, TIMBD was synthesized and purified by us as reported previously^{31 (link)}. Resveratrol and TIMBD were dissolved in dimethyl sulfoxide (DMSO) prior to treatments. The concentration of DMSO in control experiments was always 1/1000th (vol/vol) of the final medium volume. Antibodies NFĸBp-65, p-p38 MAPK, p-AKT and Lamin-B were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for p-cJUN, p-cFOS, pSTAT3, and GAPDH and appropriate anti-immunoglobulins antibodies were obtained from cell signaling (Danvers, MA, USA).

First, K562 cells were incubated for 10 minutes in solution A, which contained 1 × protease inhibitor and 1 × PhosStop cocktail (Roche, France), and then the treated cells were lysed in RIPA buffer (Thermo Fisher, Waltham, MA, USA). Control cells were lysed in RIPA buffer in the presence of 1 × protease inhibitor and 1 × PhosStop cocktail for 20 minutes. After sonication three times, the lysed cell product was centrifuged at 12000 rpm for 10 minutes at 4 °C. The protein concentration was measured using a Pierce® BCA protein assay kit (Thermo Fisher, Waltham, MA, USA). Western blotting analysis was performed as described previously^{50 (link)}. Anti-human actin, GAPDH, p38MAPK, p-p38MAPK, JNK, and p-JNK were obtained from Santa Cruz (Santa Cruz, CA, USA).