Myecl

RAW264.7 cells were cultured and stimulated with LPS with or without Hst1 (2 h pretreatment with Hst1 and then co-treatment with LPS) for the indicated times. T Protein concentration was quantified (Bio-Rad Laboratories, Hercules, CA, USA). The cells were prepared using a Nuclear Extraction Kit (Abcam, Cambridge, UK). Proteins were separated on NuPAGE 10% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA)). Nonspecific binding was blocked by soaking the membrane in a Tris-buffered saline containing 3–5% non-fat dry milk (Santa Cruz). The membranes were reacted with primary antibodies against iNOS, p-JNK, JNK, p-p38, p38, p-IκBα, p65, PCNA, and β-Actin. Horseradish peroxide-conjugated secondary antibodies were detected using enhanced chemiluminescence detection (Amersham Bioscience, Buckinghamshire, UK). Protein expression was determined by the analysis of the signals captured using an image analyzer (myECL, Thermo Scientific, Waltham, MA, USA).

Cell lysates were separated by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto nitrocellulose membranes. After blocking in 5% nonfat powdered milk in Tris-HCl buffer (TBS), the membrane was incubated with anti-HA and anti-actin monoclonal antibodies (Sigma, St. Louis, MO, USA), washed three times with TBS (50 mM Tris-HCl, pH 7.4), and probed with HRP-conjugated goat anti-mouse antibody (R & D Systems). After washing with TBS buffer with 0.05% Tween 20, the membrane was analyzed by myECL (Thermo Scientific, Waltham, MA, USA) using the enhanced chemiluminescence (ECL) reaction in the Western Chemiluminescent HRP substrate (Millipore Sigma, Burlington, MA, USA).

The cells were lysed after hypertrophy induction using buffer RIPA (Abcam, Cambridge, MA, USA) by adding a protein inhibitor cocktail (Roche Diagnostic, Indianapolis, IN, USA) to recover the extract. For the separation of proteins, the samples were denatured at 97.5°C for over 2 minutes and an SDS-PAGE at 12.5% was performed, after which the samples were transferred to a membrane of polyvinylidene fluoride (PVDF). The membrane was incubated in blocking buffer (TBS, 0.1% Tween-20 and 5% skimmed milk) for over 1 hour at room temperature with agitation. After, the membrane was incubated overnight at 4°C with agitation in TBS-Tween-20 solution at 0.1% v/v with primary antibodies against JMJD2A (1 : 2000) (Abcam, Cambridge, MA, USA, ab24545), BNP (1 : 500) (Abcam, Cambridge, MA, USA, ab19645), and ANP (1 : 500) (Abcam, Cambridge, MA, USA, ab76743). The membrane was then incubated for 1 hour at room temperature in TBS-Tween-20 solution at 0.1% v/v with the secondary antibody (1 : 3000) (Abcam, Cambridge, MA, USA, ab6721). Lastly, the membrane was treated with Luminata-HPR Substrate (EMD Millipore) for 5 minutes at room temperature and was visualized by chemiluminescence using MyECL (Thermo Scientific, MA, USA).