Hrp conjugated goat anti rabbit antibody

RIPA Buffer was used to extract total proteins from ovaries. The BCA Protein Assay Kit was employed to detect the concentration of protein. Equivalent amounts of total protein were subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to PVDF membranes. After blocking with skim milk, the membranes were incubated overnight with an anti-GAPDH, anti-ASK1, anti-p-ASK1, anti-JNK, anti-p-JNK, anti-Bcl-2, anti-Bax, anti-caspase-9/3, anti-Cyt-c, anti-OPA1, anti-Mfn1, anti-Mfn2, anti-Drp1, anti-Fis1, or anti-PGC1a antibody (Table 2). After that, the membranes were incubated with a secondary HRP-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The Enhanced Chemiluminescence Kit (Thermo Fisher Scientific) was used to visualize the proteins. Alpha View Software (Cell Biosciences, Preston VIC, Australia) was used for densitometric analysis.

The expression of GGT in the tumor was confirmed by performing immuno-histochemical staining for GGT on tumor tissue excised from xenograft tumor models. Briefly, the tissues were rinsed in PBS twice and fixed in 10% buffered formalin for 24 h. Paraffin embedding and sectioning (3 μ) was done. Sections were probed for the expression of GGT by incubating with primary rabbit anti-GGT1 polyclonal antibody (Abcam, Dilution 1:100) at 4°C overnight. Detection of Primary antibody was performed using HRP-conjugated goat anti-rabbit antibody (Santa Cruz, Dilution (1:500). Diaminobenzidine (DAB) was used as a chromogen and sections were counter stained with Harris’s hematoxylin.

HEK293T cells were purchased from American Type Culture Collection (Manassas,VA), cultured in Dulbecco’s modified Eagle’s media (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Korea, Seoul, Korea), and transfected with plasmid using Lipofectamine 2000 (Thermo Fisher Scientific Korea) according to the manufacturer’s instructions. Subsequently, the cells were lysed with M-PER mammalian protein extraction reagent (Thermo Fisher Scientific Korea) at 48 h post-transfection, and processed for Western blotting as described previously [27 (link)]. The antibodies used were anti-FLAG antibody (1:2000, Sigma-Aldrich, catalog number F1804), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (1:2000, Trevigen, Gaithersburg, MD, 2275-PC-100), HRP-conjugated goat anti-mouse antibody (1:4000, Santa Cruz Biotechnology, Dallas, TX, sc-2005), and HRP-conjugated goat anti-rabbit antibody (1:4000, Santa Cruz Biotechnology, sc-2004). Band intensity on the Western blots was analyzed using ImageJ.

The cardiomyocytes were collected, and nucleoproteins were extracted using a nuclear protein extraction kit (Merck Millipore, Darmstadt, Germany). Nucleoproteins were separated by electrophoresis on 6/12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). After blocking for 1 h with 5% bovine serum albumin, these PVDF blots were probed with rabbit polyclonal antibodies against brain natriuretic peptide (BNP) (Abcam, 1:1000 dilution), atrial natriuretic peptide (ANP) (Abcam, 1:5000 dilution), lysine 9-acetylated histone H3 (H3K9ac) (Abcam, 1:5000 dilution), beta-myosin heavy chain (β-MHC) (Abcam, 1:5000 dilution), PCAF (Abcam, 1:500 dilution), and P300 (Abcam, 1:2000 dilution), rabbit polyclonal antibodies against JNK and phospho-JNK (p-JNK) (Cell Signaling, 1:1000 dilution), or rabbit polyclonal antibodies against β-actin and histone H3 (Beyotime, 1:5000 dilution). All antibodies were diluted in tris-buffered saline with tween 20 (TBST) containing 5% non-fat milk, and incubations were performed at 4°C overnight. HRP-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, 1:2000 dilution) was used as the secondary antibody. After scanning, the bands were subjected to analysis using the Quantity One (Version 4.4) software package (Bio-Rad, CA, USA).

Supernatants were harvested from transient transfected Expi293F culture and analyzed by electrophoresis on a 12% polyacrylamide gel using Coomassie brilliant blue staining method according to the Laemmli35 (link). Western blotting was also analyzed based on Sambrook et al.36 using a semidry blotting system (Bio-Rad, USA). Polyclonal rabbit anti-t-PA antibody (1/1000 dilution, Abcam, USA) and HRP conjugated goat anti-rabbit antibody (1/2500 dilution, Santa Cruz. USA) were used as primary and secondary antibodies, respectively. Ultimately, protein bands were visualized by adding DAB solution (Sigma-Aldrich, Germany).