Qproteome kit

A Qproteome Kit (Qiagen, Hilden, Germany) was used to separate and extract cytosolic or membrane proteins from HEK293 cells post transfection in accordance with the manufacturer’s guidelines. Proteins were then homogenized by brief sonication, and protein concentration was determined using BCA Protein Assay Kit (Pierce Biotechnology, IL, USA). Cell lysates were mixed with NuPAGE ® LDS sample buffer (Invitrogen CA, USA) and dithiothreitol (Sigma, MO, USA), incubated at 95 °C over 5 min, and then cooled on ice for 5 min. Proteins were separated by SDS-PAGE (4–12% NuPAGE Bis-Tris Gels, Invitrogen) and then transferred to nitrocellulose membranes and membranes blocked with 5% skim milk (in TBST) for 1 h at room temperature. GFP monoclonal mouse antibody (dilution 1:2000, Roche (11814460001), Basel, Switzerland), MT-CO2 (dilution 1:2000, Abcam (ab3298), Cambridge, UK) and GAPDH (dilution 1:5000, Cell Signaling (D16H11), MA, USA) were used to probe membranes overnight at 4 °C. Blotted membranes were washed 3 × 10 min with TBST and incubated with the relevant secondary antibody conjugated to HRP. After another 3 × 10 min with TBST, proteins were detected with ECL Plus Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) on a LAS-3000 (Fujifilm, NSW, Australia). Quantitation was performed using the Multi-Gauage 3.11 Software (Fujifilm).

Histone lysates were extracted using the Qiagen Qproteome kit according to the manufacturer’s protocol and quantified using a Bradford assay. Twenty ug of histone lysate was loaded onto 4%–20% Novex Tris-glycine gel (Invitrogen) and transferred to a nitrocellulose membrane. Membranes were blocked in Odyssey buffer (LiCor Biosciences) and incubated overnight with primary antibodies. The following antibodies were used for Western blots according to the manufacturer’s protocol: histone subunit H3 (Santa Cruz Biotechnologies, #sc-8654), H3K9ac (Cell Signal Technologies, #9671s), H3K14ac (Millipore, #07-353), H3K4me3 (Epigentek, #A-4033), and H3K27me3 (Millipore, #17-622). After overnight incubation, membranes were incubated with secondary fluorescent antibodies according to the manufacturer’s protocol (LiCor Biosciences) and visualized using the Odyssey Infrared Imaging System (Licor Biosciences). The total intensity of each band was divided by the total intensity of histone subunit H3 and presented as a percent change relative to the average of saline controls. For CCx, A total of 6 animals per group were assessed on 2 separate membranes. For HC, a total of 3 animals per grouped were assessed on 2 separate membranes. Membranes were stripped four times using stripping buffer (LiCor Biosciences) between incubations with primary antibody.