Anti ampk

Western blot analysis was carried out as described by Misiura et al. [30 (link)]. The membranes were incubated with primary antibodies diluted 1000 times in 5% bovine serum albumin (Sigma Aldrich, Saint Louis, MO, USA) in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6). Anti-PARP, anti-AMPK and anti-caspase-7 and anti-GAPDH, were purchased from Cell Signaling Technology, Danvers, MA, USA; anti-PRODH/POX from St John’s Laboratory, London, UK), followed by incubation with alkaline phosphatase-linked goat anti-rabbit or anti-mouse antibodies (dilution: 1:10,000 in 5% non-fat dried milk (Santa Cruz Biotechnology, Dallas, TX, USA) in TBS-T; Sigma Aldrich, Saint Louis, MO, USA). The bands’ intensities were semi-quantitatively measured in ImageJ software (https://imagej.nih.gov/ij/, accessed on 27 October 2021). All experiments were run at least in triplicates.

Baicalin (purity ≥ 98%) was obtained from Xi’an Kai Lai Biological Engineering Co., Ltd. (Xi’an, China). Fluoxetine hydrochloride (Flu) was purchased from Changzhou Siyao Pharmaceuticals Co., Ltd. (Changzhou, China). Lipopolysaccharide (LPS), 4,6-diamidino-2-phenylindole (DAPI), and compound C were bought from Sigma-Aldrich Co (St. Louis, USA). TAK-242 (a TLR4 antagonist) and LY294002 (a PI3K inhibitor) were products purchased from Apex Bio (Houston, USA). Poly-d-lysine was obtained from Sigma (MO, USA). Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were supplied by Elabscience Biotechnology Co., Ltd. (Wuhan, China). The antibodies were obtained from the cited commercial sources: anti-p-PI3K (#4228), anti-PI3K (#4292), anti-p-Akt (Ser473, #9271), anti-Akt (#9272), anti-β-actin (#4967), anti-PCNA (#13110), anti-p-AMPK (#2531), and anti-AMPK (#2603) were from Cell Signaling Technology (Beverly, MA, USA); anti-PTEN (sc-7974) and anti-p-PTEN (sc-377573) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-TLR4 (AF7017), anti-p-GSK3β (AF2016), and anti-GSK3β (AF7814) were from Affinity Biosciences (Changzhou, China); and anti-FoxO1 (ab52857), anti-p-FoxO1 (Ser 256, ab131339), and goat anti-rabbit IgG H&L (Alexa Fluor® 488, ab150077) were from Abcam (Cambridge, MA, USA).

Anti-FLAG M2 (Cat# F1804), tubulin (Cat# T5168) and actin (Cat# A2066) antibodies were obtained from Sigma-Aldrich. The rabbit anti-ATGL antibody was obtained from Cayman Chemicals. Anti-p53 (DO-1) was purchased from Santa Cruz Biotechnology. Anti-Bcl-XL (Cat# 2762S), Anti-AMPK (Cat# 2532) and phospho-AMPK (Cat# 2535S) were obtained from Cell Signaling. The rabbit polyclonal antibody against G0S2 has been described previously [44]. Whole-cell extracts were obtained by harvesting cells and boiling in 1X Laemmli buffer. Western blotting was performed using standard protocols for SDS-PAGE and wet transfer onto nitrocellulose membranes (Bio-Rad).

Dulbecco's modified Eagle's medium (DMEM) and other culture reagents were obtained from Gibco Life Technologies (Grand Island, NY).The cell culture plates were purchased from Nalge Nunc International (Roskilde, Denmark). Human insulin (HumulinR) was from Eli Lilly S.A.S.(Fegersheim, France). Bovine serum albumin (BSA), forskolin, IBMX, and dexamethasone were purchased from Sigma (St Louis, MO, USA). Compound C was purchased from Calbiochem (San Diego, CA). Anti-CREB, anti-phospho-CREB (Ser133), anti-PPARγ, anti-C/EBPα, anti-fatty acid synthase (FAS), anti-fatty acid binding protein 4 (FABP4), anti-C/EBPβ, anti-AMPK, anti-phospho-AMPK (Thr172), anti-acetyl-CoA carboxylase (ACC), anti-phospho-ACC(Ser79), anti-β-actin, anti-α1-tubulin, anti-mouse IgG and anti-rabbit IgG conjugated with horseradish peroxidase were from Cell Signaling Technology (Beverly, MA, USA). Murine-derived 3T3-L1 preadipocytes were purchased from American Type Culture Collection (Rockville, MD). Berberine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

The tissue was harvested from the kidney cortex of the sacrificed animals. Protein was extracted for western blotting from the cortex. The primary antibodies used in this study were Fatty Acid and Lipid Metabolism Antibody Sampler Kit (#8335), anti-TNFR1 (#13377), anti-AMPK (#5832), and anti-p-AMPK (#2535), from Cell Signaling Technology (Danvers, MA, USA); anti-SREBP-1 (sc-13551) and anti-SREBP-2 (sc-13552), from Santa Cruz Biotechnology (Dallas, TX, USA); anti-CD36 (NB400-144ss), anti-β actin (NB600-501), and anti-α tubulin (NB100-690), from Novus Biologicals (Littleton, CO, USA); anti-PPARα (GTX01098), anti-HDAC1 (GTX100513), and anti-histone H3 (GTX122148), from GeneTex (Irvine, CA, USA); and anti-KIM 1 (ab190696, Abcam, Cambridge, UK). The membrane was then subjected to the secondary antibody, either anti-mouse IgG or anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA), which was dissolved in 5% skim milk in TBST for 1 h. Next, the membrane was incubated for 1–2 min in enhanced chemiluminescence mixture (JT96-K004M, T-Pro Biotechnology, Zhonghe, New Taipei City, Taiwan) for visualization. The western blotting assay was repeated at least three times.

Western blot analysis was performed, as previously described [24 (link)]. Initial cell lysis was used by RIPA buffer and protease/phosphatase inhibitors. Equal amounts of protein samples (25 or 50 µg) were separated to sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membranes by electrophoresis. The membranes were washed with 0.1% Tween 20 in TBS buffer (TBST) and were reactivated in blocking solution (5% bovine serum albumin (BSA)). The membranes were then incubated with anti-AMPK (Cell signaling; Danvers, MA, USA, 2532), phospho-AMPK (Cell signaling; 2535), brain-derived neurotrophic factor (BDNF, Abcam; Cambridge, UK, ab108319), tropomyosin receptor kinase B (TrkB, Cell signaling; 4603), phospho-TrkA (Tyr785)/TrkB (Tyr816) (Cell signaling; 4168), cAMP response element-binding protein (CREB, Cell signaling; 9197), phospho-CREB (Ser133) (Cell signaling; 9198), or β-actin (Santa Cruz Biotechnology; Dallas, TX, USA, sc-47778HRP) overnight at 4 °C. All primary antibodies except β-actin (1:5000 dilution) were used at a 1:1000 dilution. After repeated washing times with TBST, the membranes were reacted with HRP-conjugated secondary antibodies (1:5000 dilution). Protein detection was performed using an ECL reagent (Bio-Rad Laboratories, Hercules, CA, USA) and captured by Solo6S EDGE (Vilber Lourmat Sté, Collégien, France).