Anti suv39h1

Co-immunoprecipitation (Co-IP) was performed according to a previously described method^{18 (link)}. Briefly, mouse cardiac muscle or primary rat neonatal cardiomyocytes lysates were incubated with anti-Kindlin-2 (Millipore, Billerics, MA, USA MAB2617, clone 3A3) and anti-SUV39H1 (Cell Signaling Technology, Danvers, MA, USA) antibodies at 4 °C overnight followed by incubation with protein A/G agarose beads (Santa Cruz Biotechonology). After that, the beads were washed three times with NP40 buffer, the bound proteins were eluted with 2 × SDS loading buffer and then boiled at 100 °C for 5 min. Precipitated proteins were resolved by 10% SDS–PAGE and subjected to western blotting analysis. Western blotting were performed by using anti-SUV39H1 (Cell Signaling Technology, Danvers, MA, USA), anti-Kindlin-2 (Millipore, Billerica, MA, USA) antibodies, anti-GATA4 (G-4, Santa Cruz Biotechnology), anti-GATA5 (55433-1-AP, Santa Cruz Biotechnology), anti-GATA6 (YT1885, ImmunoWay Biotechnology) and anti-GAPDH (Santa Cruz Biotechnology). Secondary antibodies were goat anti-mouse HRP and goat anti-rabbit HRP (both Santa Cruz Biotechnology, Inc.).

ChIP was performed as previously described (24 (link)), with the Pierce agarose ChIP kit (Thermo Scientific). For ChIP, anti-RNAP II (17-672 [Millipore] or sc-899 [Santa Cruz]), anti-EZH2 (17-662; Millipore), anti-EED (17-10034; Millipore), anti-SUZ12 (17-661; Millipore), anti-Jarid2 (AB192252; Abcam, Inc.), anti-histone H3 (AB1791; Abcam, Inc.), anti-histone H3K27me3 (AB6002; Abcam, Inc.), anti-EHMT2 (3356S; Cell Signaling), anti-SUV39H1 (8729S; Cell Signaling), anti-KDM1 (LSD1) (2139S; Cell Signaling), anti-trimethyl histone H3 (Lys9) (AB8898, Abcam, Inc.), and anti-dimethyl histone H3 (Lys9) (AB1220; Abcam, Inc.) antibodies were used. The percentage-of-input method was used to calculate the enrichment of proteins in specific regions of the HIV-1 genome.

After primary rat neonatal cardiomyocytes were fixed with 4% paraformaldehyde solution at room temperature for 15 min, they were treated with 0.5% Triton X-100 at 37 °C for 5 min and blocked with 5% BSA at room temperature for 1 h. The cells were then incubated with 1:200 dilution of anti-Kindlin-2 (Millipore) and dilution of 1:200 anti-SUV39H1 (Cell Signaling Technology) and then with a 1:100 dilution of Alexa Fluor 568-conjugated IgG (Invitrogen) or with a 1:100 dilution of Alexa Fluor 488-conjugated IgG (Invitrogen) for 1 h at room temperature and incubated with DAPI for 2 min for the detection of the nuclei. Images were captured with a TCS SP5 confocal microscope (Leica, Germany).