Anti pdk1 antibody

For western blotting, 20 μg of cellular protein extracts were used [23 (link)]. We used rabbit anti-Akt antibody, anti-S473- and anti-T308-phospho-specific Akt antibodies, anti-PDK1 antibody, anti-S241-phospho-specific PDK1 antibody, anti-JNK antibody, anti-T183/Y185-phospho-specific JNK antibody, anti-p38 antibody, anti-phospho-specific p38 antibody (Cell Signaling Technology, USA), and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA). Relative protein amounts were evaluated using Chemidoc XRS system (Bio-Rad, USA) and ImageJ tool (National Institute of Health, USA).

Western blotting was performed as previously described [12 (link)]. Briefly, total cell lysates of non-PAH-PASMCs and PAH-PASMCs were extracted in commonly used radioimmunoprecipitation buffer with 10 µg/mL phenylmethylsulfonyl fluoride (Sigma) and then concentrated by centrifugation at 12,000 rpm for 20 min. Protein samples (7 µg) were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel and blotted onto nitrocellulose membranes. Blots were incubated with anti-PDK1 antibody (1:1000; #3820; Cell Signaling Technology, Danvers, MA, USA), anti-PDH phospho S293 (1:1000; ab177461, Abcam, Cambridge, UK), anti-PDH (1:1000; ab110330, Abcam), and anti-beta actin (1:1000; ab6276, Abcam). The secondary antibody was horseradish-peroxidase-conjugated anti-mouse (1:10,000; NA931, GE Healthcare, Chicago, IL, USA) or anti-rabbit (1:10,000; NA934, GE Healthcare) IgG antibody. Positive signals were detected by a chemiluminescence system (ECL plus, GE Healthcare Bio-Sciences, Piscataway, NJ, USA).