Anti flag antibody

Immunoblotting was performed as described previously.14 (link),15 (link) Whole cell lysates were extracted and equal amounts of total proteins were subjected to SDS-PAGE separation, followed by immunoblotting with specific antibodies. The primary antibodies PARP, Bcl-2, Bim, phospho-NF-κB p65 (p-p65) (Ser536), NF-κB p65, phospho-IκBα (p-IκBα) (Ser32), IκBα, phospho-IKKα/β (p-IKK) (Ser176/180), IKKα, IKKβ, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Flag antibody was purchased from Medical & Biological Laboratories (Tokyo, Japan). Anti-mouse and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugated antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

HDL fractions (10 μL) were separated using 10% SDS‐PAGE. After transfer, nitrocellulose membranes were probed with anti‐ANGPTL4 antibody 1/1000 (Abcam, Cambridge, UK) overnight and then incubated with horseradish peroxidase–conjugated IgG secondary antibody 1/3000 (Amersham Biosciences, Piscataway, NJ) for 1 hour. Signals were detected by ECL chemiluminescence (Thermo Fisher Scientific, Rockford, IL).
To further confirm the presence of ANGPTL4 on HDL fractions, HDLs (400 μL) and lysis buffer (400 μL) were incubated with anti‐ANGPTL4 antibody (Santa Cruz, Dallas, TX) or IgG (Cell Signaling Technology, Beverly, MA) coupled with protein G plus/protein A‐agarose (Santa Cruz, Dallas, TX) overnight. After washing, the immunoprecipitated proteins were processed for Western blotting as described above.
HEK 293 cells transfected with plasmid containing EL cDNA (GFP‐EL‐flag) were lysed in 1 mL ice‐cold lysis buffer (10 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L benzamidine, and 0.5% Triton X‐100 [pH 7.4]). The lysates were analyzed by Western blotting with anti‐flag antibody (Medical and biological laboratories, Nagoya, Japan) and anti‐GAPDH antibody (Santa Cruz, Dallas, TX).