Anti angptl4 antibody

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).

Cells were harvested in sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of proteins were separated by using 10% SDS-PAGE system, transferred onto the polyvinylidene fluoride membrane (Millipore, USA). The membranes were blocked with 5% non-fat dried milk. Then, the membrane was blotted with the primary antibodies: anti-ANGPTL4 antibody (1:1000, Santa Cruz, USA), the polyclonal rabbit anti-HA antibody (1:1000, MBL, Japan), Anti-AKT and anti-phospho-AKT (Ser473) (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH antibody (1:3000, ZSGB-BIO, China), diluted with 1% non-fat dried milk in TBST. After washed with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000, ZSGB-BIO, China). The membranes were washed with TBST and scanned by ChemiDoc XRS+ chemiluminescence imaging system (Bio-Rad, USA). Each examination was tested in triplicate, and GAPDH was served as the internal reference.