Anti dykddddk tag mab

Transfected cells were prepared in Laemmli buffer (Bio-Rad Laboratories) containing 2.5% 2-mercaptoethanol (2-ME). Proteins were separated on either 10% or 12% SDS–PAGE gels and transferred onto Immobilon-P membranes (Merck Millipore). The membranes were first probed with the appropriate primary antibodies. An anti-HIS tag monoclonal antibody (mAb), anti-HA tag mAb (both at a 1:2.5 × 10³ dilution) (Medical & Biological Laboratories Co.), anti-DYKDDDDK tag mAb (1:2 × 10³ dilution; for detecting the FLAG tag) (Fujifilm Wako Pure Chemical Co.), and anti-ß-tubulin antibody (1:10³ dilution; as the loading control) (Abcam) were used. The membranes were subsequently incubated with goat horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG (both 1:2 × 10⁴ dilution; Thermo Fisher Scientific) as secondary antibodies. Proteins were visualized by enhanced chemiluminescence using SuperSignal West Dura substrate (Thermo Fisher Scientific) with an ImageQuant LAS 4000 system (GE Healthcare Life Sciences).

Anti-MyD88 Ab, anti-phospho IRAK4 Ab, anti-IRAK4 mAb, anti-phospho p65 Ab, anti-phospho ULK1 mAb, anti-ULK1 mAb, anti-phospho AMPKα mAb, anti-AMPKα mAb, anti-LC3B mAb and horseradish peroxidase (HRP)-conjugated Rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-DYKDDDDK tag mAb, anti-tag antibody magnetic beads, DYKDDDDK peptide, coomassie G-250, compound C and HRP-conjugated anti-β-actin Ab were obtained from WAKO Pure Chemical Industries (Osaka, Japan). Lipopolysaccharide (LPS, Salmonella enterica serotype typhimurium) was purchased from Sigma Aldrich (St. Louis, MO, USA). Pam3CSK4, FLA-ST standard, FSL-1, ssRNA40/LyoVec and poly (I:C) HMW were obtained from InvivoGen (San Diego, CA, USA). Halt protease & phosphatase inhibitor single-use cocktail was purchased from Thermo Scientific. RIPA lysis buffer was obtained from Merck Millipore (Billerica, MA, USA). 3-methyladenine was purchased from Santa cruz Biotechnology (Dallas, TX, USA).