Phosphorylated nf κb

TLR2 and phosphorylated NF-κB proteins were isolated from the cytoplasmic extraction of Pam₃CSK₄-stimulated Raw264.7 cells using Proprep buffer, and subsequently detected using antibodies specific for TLR2 (1:2000, Cell Signaling Technology, Danvers, MA, USA), phosphorylated NF-κB (1:1000, Cell Signaling Technology, Danvers, MA, USA), and β-actin (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) as described previously [29 (link)]. Cultured cells were pretreated with 20 µM of CU-CPT22 and phloretin for 1 h, and then stimulated with 1 µg/mL Pam₃CSK₄. We measured the concentration of protein on a Nanodrop spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA; range: 0.06–820 mg/mL bovine serum albumin). β-actin was used as the control in the Western blot. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the relative intensities of the protein bands detected using these antibodies.

Lysis buffer was used to extract total protein from colon tissues, and BCA Protein Assay Kit (Applygen Technologies Inc., Beijing, China) was put into use to detect protein concentrations. Primary antibodies including β-actin (1:1000), NF-κB (1:1000) and phosphorylated (p)-NF-κB (1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). In addition, the primary antibody of Hsp70 (1:1000) was obtained from Abcam (Cambridge, UK). Blots were incubated with horseradish peroxidase-coupled secondary antibody, and were finally quantified by ImageJ software (NIH, Bethesda, MD, USA).