Goat anti rabbit igg h l hrp

Cells were lysed with RIPA buffer (Tech & Innovation, China) containing 0.5 mM EDTA solution and a protease & phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) to release proteins. Proteins were then loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membranes. Immunoblots were probed with primary antibodies against p-NF-κB p65, p-p38, p-ERK1/2, p-JNK (Cell Signaling Technology, USA) and α-tubulin (Abcam, UK), followed by incubation with secondary antibodies against goat anti-rabbit IgG (H+L)-HRP (GenDEPOT, USA). A Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) was used to detect protein bands. Signal intensity was determined with a ChemiDoc XRS+ imaging system (Bio-Rad, USA).

RAW 264.7 cells (2 × 10⁵ cells/well) were treated with CFE or positive drugs before LPS stimulation. Western blot analysis was performed by extracting total protein using RIPA lysis buffer (Tech & Innovation, China) with an EDTA solution and protein inhibitor cocktail (Thermo Fisher Scientific, USA). The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to evaluate the protein content. Protein (30 μg) was separated using 10% SDS-PAGE before being transferred to PVDF membranes. After transfer, the membranes were immobilized with 5%skimmed milk in TBST buffer and placed with primary antibodies against p-NF-κB65, p-p38, p-JNK, p-ERK1/2 (Cell Signaling Technology, USA), and α-tubulin (Abcam, UK) before being immediately followed by incubation with secondary antibodies against goat anti-rabbit IgG(H+L)-HRP (GenDEPOT, USA). The detection of protein bands was performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific), and the signal intensity was determined with the ChemiDoc XRS+ imaging system (Bio-Rad, USA).

Western blot was performed as previously reported [14 ]. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Gendepot). The membrane was incubated following primary antibodies: p-AMP-activated protein kinase (AMPK), AMPK (Cell signaling, Beverly, MA, USA), sterol regulatory-element binding proteins (SREBP)1c (Santa Cruz Biotechnology, Dallas, TX, USA), fatty acid synthase (FAS; Cell signaling), peroxisome proliferator-activated receptor-γ (PPARγ; Cell signaling), CCAAT/enhancer-binding protein-α (C/EBPα; Cell signaling), SREBP1 (Abcam), SREBP2 (ABclonal, Wuhan, Hubei, China), cytochrome P450 family 7 subfamily a member 1 (CYP7A1; ABclonal), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMCGR; Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology). The secondary antibodies, including Goat anti-Mouse IgG (H+L) and Goat anti-Rabbit IgG(H+L)-HRP, were obtained from Gen-depot. The blots were visualized using the West-Q Femto Clean enhanced chemiluminescence (ECL) solution (Gendepot) and LuminoGraph III Lite (ATTO, Tokyo, Japan), following the manufacturer’s instructions. Quantitative analysis was measured with CS Analyzer 4 (ATTO).

In this experiment, cells were pretreated with different concentrations of lipid fractions of A. japonicus. Then, these cells were stimulated with or without 1 μg/mL of LPS for 24 h. Proteins were extracted from these cells by using RIPA buffer (Tech & Innovation, Hebei, China). Protein concentrations were determined with Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (30 µg) were electrophoresed separately by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then they were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with primary antibodies that specifically identified the following transcription factors: p-NF-κB, p65, p-p38, p-JNK, and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), and α-tubulin (Abcam, Cambridge, UK). After washing the membrane, the bolts were incubated with Goat Anti-Rabbit IgG (H + L)-HRP (GenDEPOT, Katy, TX, USA) at 37 °C for 1 h. The bands were visualized with the following tools: Pierce^® ECL Plus Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA), ChemiDoc XRS+ imaging system, and ImageLab software (Bio-Rad, Hercules, CA, USA).

The Western blot assay was carried out as described by Monmai, C. et al. [57 (link)]. The concentrations of proteins diluted 1:40 in lysis buffer were measured using Bradford reagent (Welgene) and determined from a standard curve of bovine serum albumin (BSA; Sigma-Aldrich) (Figure S1). The following primary antibodies were incubated at 4 °C overnight: PPARγ (1:2000; Cell Signaling, Danvers, MA, USA), C/EBPα (1:2000; Cell Signaling), p-ERK 1/2 (1:2000; Cell Signaling), ERK 1/2 (1:2000; Cell Signaling), p-Akt (1:2000; Cell Signaling), Akt (1:2000; Santa Cruz Biotechnology, Dallas, TX, USA), p-NF-κB p65 (1:2000; Cell Signaling), NF-κB p65 (1:2000; Santa Cruz Biotechnology), and GAPDH (1:5000; Santa Cruz Biotechnology). Secondary antibodies were incubated at room temperature for 2 h [goat anti-rabbit IgG(H + L)-HRP (1:5000; GenDepot, Baker, TX, USA) or m-IgGκ BP-HRP (1:5000; Santa Cruz Biotechnology)]. Protein signaling was detected using Clarity Max™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA). The detected signaling was imaged and quantified using a ChemiDoc Imaging System (Bio-Rad).

Cell lysates were prepared using RIPA buffer (Tech & Innovation, Hebei, China, Cat# BRI-9001) containing 0.5 mM EDTA solution, and a protease & phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA, Cat# 78440). SDS-PAGE and western blotting were performed. The protein was analyzed by immunoblot using primary antibodies against phospho-nuclear NF-κB-p65 (Cell Signaling Technology, MA, USA, Cat# 3033, RRID: AB_331284), phospho-p38 (Cell Signaling Technology, MA, USA, Cat# 9211, RRID:AB_331641), phospho-ERK1/2 (Cell Signaling Technology, MA, USA, Cat# 9101, RRID: AB_331646), phospho-JNK (Cell Signaling Technology, MA, USA, Cat# 9251, RRID: AB_331659), and α-tubulin (Abcam, Cambridge, UK, Cat# ab15246, RRID:AB_301787), and then was incubated with secondary antibodies as goat anti-rabbit IgG (H+L)-HRP (GenDEPOT, TX, USA, Cat# SA006-500). The protein bands were measured by the ChemiDoc XRS+ imaging system, and ImageLab software (Bio-Rad, Hercules, CA, USA). Results were presented as triplicate independent experiments.