Ecl detection kit

Whole proteins from cultured cells were extracted by using protein extraction kit (Nanjing Kaiji, Nanjing, China). Equal amounts of proteins (50 μg) from each sample were resolved in 8% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Non-specific binding sites in the membranes were blocked with 5% non-fat dry milk in incubation buffer before addition of primary antibodies (GAPDH, VEGF, c-Fos, HIF-1α, ERK and pERK, 1:500∼1:2000). Blots were washed and incubated with appropriate HRP-conjugated secondary antibodies. Protein bands were visualized by using an ECL detection kit (Amersham Pharmacia, Uppsala, Sweden). Protein expression was determined by using Quantity One software 4.5.0 and was normalized to GAPDH.

The anti-TDP-43 antibody was described previously,^{66 (link)} and the following additional primary antibodies were used: anti-iNOS (Abcam, Cambridge, MA, USA), anti-COX-2 (Epitomics, Burlingame, CA, USA), anti-GAPDH (Chemicon, Temecula, CA, USA), anti-phospho-MEK (S217/221) (Cell Signaling Technology, Beverly, MA, USA), anti-MEK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-ERK (Santa Cruz Biotechnology), anti-ERK (Santa Cruz Biotechnology), anti-phospho-JNK (Epitomics), anti-JNK (Santa Cruz Biotechnology), anti-phospho-p38 (Epitomics), anti-p38 (Santa Cruz Biotechnology), anti-phospho-c-Raf (S338) (Cell Signaling Technology), anti-c-JUN (Proteintech, Chicago, IL, USA), anti-phospho-Glycogen Synthase (S641) (p-GS) (Epitomics) and anti-MAP2 (Santa Cruz Biotechnology). The secondary antibodies used included horseradish peroxidase-conjugated sheep anti-mouse and anti-rabbit antibodies (Amersham Pharmacia Biotech, Peapack, NJ, USA). The antibody-bound proteins were visualized using an ECL detection kit (Amersham Biosciences, Piscataway, NJ, USA) or Alexa Fluor 488 (green) donkey anti-mouse IgG (Invitrogen, La Jolla, CA, USA).

Cells were lysed and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked and incubated with primary antibodies (Table S2) for 1 h at room temperature with agitation, followed by incubation with secondary antibodies (1:2000; Santa Cruz). Then, the membranes were developed using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). A rabbit polyclonal β-actin antibody (sc1616-R, 1:200; Santa Cruz) was used as a protein loading control. The intensity of the protein bands was determined via densitometry using Image J system.

COCs were denuded and GV oocytes (50 per sample) were washed three times in PVA–PBS, and then resuspended in extraction buffer (PRO-PREP; Intron Biotechnology, Seoung, Korea). The extracted proteins were separated by 10% (w/v) SDS–PAGE using a Bio-Rad apparatus (Bio-Rad) and then electrophoretically transferred to PVDF membranes, employing a Bio-Rad Mini Trans-Blot Cell. The membrane was blocked with 5% (w/v) skim milk and 0.5% (v/v) Tween-20 in Tris-buffered saline and subsequently exposed to primary antibody directed against CDK9 at 1:1000 dilution. The antibody solution was prepared in Tris-buffered saline, containing 5% (w/v) nonfat dry milk powder and 0.1% (v/v) Tween-20. The membrane was next washed in Tris-buffered saline with 0.5% (v/v) Tween-20 for 15 min and antibody–antigen complexes were detected using anti-rabbit IgG peroxidase conjugates (Abcam, ab6721, Cambridge, MA, USA), followed by employment of an ECL detection kit (Amersham Bioscience).

Western blot was performed as described previously [22 (link)]. Left ventricles were homogenized, and the cells were directly lysed in ice-cold RIPA buffer. The samples were analyzed by SDS-PAGE. Transfer the protein to a polyvinylidene fluoride microporous membrane (Bio-Rad) with primary antibodies PFKFB3 (Abcam, USA; 1: 1000), HIF1α (CST, USA; 1: 1000), and anti-GAPDH (internal control; Kangcheng Co., Ltd., China; 1 : 8000). The bands were detected by horseradish peroxidase-coupled secondary antibodies (Cell Signaling Technology, 1 : 6000) and were visualized using the ECL detection kit (Amersham Pharmacia Biotech) and quantified with a video documentation system (Gel Doc 2000, Bio-Rad).

Isolated protein samples from aortic homogenates were separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The proteins (70 μg) were then transferred to polyvinylidene difluoride membranes and incubated with primary monoclonal mouse anti-PPARβ (1:700 dilution), anti-eNOS (1:1000 dilution) and anti-iNOS (1:1000 dilution) antibodies overnight, and then with the corresponding peroxidase-conjugated secondary antibodies (1:1000 dilution) on the second day. Antibody binding was detected using an ECL detection kit (Amersham Biosciences, Piscataway, NJ, USA) and densitometric analysis was performed using a quantitative imaging system (Bio-Rad, USA). All western blotting experiments were repeated three times.

Equal amounts of total protein lysates (40 μg per lane) were resolved by sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis and transferred to polyvinylidene difluoride (PVDF) blots. The blots were blocked and immuno-blotted with the primary antibodies overnight, following by incubation with the corresponding secondary antibodies. An enhanced chemiluminescence (ECL) detection kit (Amersham, Buckinghamshire, UK) was applied to detect targeted protein bands. The ImageJ software from NIH was utilized for data quantification.