Anti irak m

Macrophages were infected with Mtb for the indicated times. Cells were lysed with lysis buffer consisting of 10 mM Tris–HCl, 1 mM EDTA, 140 mM NaCl, 0.1% DOC, 0.1% SDS, and Triton X-100 containing complete protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). Western blotting was performed as previously described (27 (link)). Anti-phospho-GSK3β, anti-IκBα, anti-cathepsin D, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-PI3K, anti-PI3K, anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, anti-phospho-MEK1/2, anti-phospho-JNK1/2, anti-phospho-IκBα, and anti-IRAK-M were purchased from Cell Signaling Technology.

THP-1 or HepG2 cells treated with aLTA were lysed with 2× reducing buffer and boiled for 5 min at 100 °C. Samples were loaded and resolved in 10% or 12% SDS-PAGE gels and proteins were transferred onto polyvinylidene fluoride (PVDF) membranes overnight at 40 V. The membranes were blocked with 5% bovine serum albumin (BSA) or skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature (RT). After washing three times with TBST, membranes were incubated with anti-human C3, anti-human C5, anti-TLR2, anti-β-actin, anti-p65 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti- Interleukin 1 Receptor Associated Kinase (IRAK) 2, anti-IRAK-M, anti- suppressor of cytokine signaling (SOCS)-1 or anti-phospho p65 (Cell Signaling Technology Inc., Danvers, MA, USA) primary antibodies diluted in TBST (1:1000) for 2 h at RT and then washed three times with TBST. The membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibody (1:2000 in TBST) for 2 h at RT. After washing three times with TBST, the membranes were treated with enhanced chemiluminescence (ECL) reagent and exposed to X-ray film. β-actin was used as the internal loading control.