Rabbit anti p27 antibody

Human cardiac myocytes were transfected with 1 μg of WT and variant plasmid DNA. Then, we harvested the cells 48 h after transfection. We used western and immunoprecipitation lysis buffer (Beyotime, Shanghai China) with protease and phosphatase inhibitor cocktail (Beyotime) to lysis cells. The proteins were subjected to 10% SDS/PAGE and were then transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA) and immunostained with rabbit anti‐NRDG4 antibody (dilution 1 : 500; Novus Biologicals, Centennial, CO, USA), rabbit anti‐GAPDH antibody (dilution 1 : 5000; Proteintech, Chicago, IL, USA) rabbit anti‐P27 antibody (dilution 1 : 1000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. The membranes were incubated with HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG(H + L) secondary antibody (dilution 1 : 10 000; Proteintech) [21, 22]. All the raw immunoblots are available in the supplement (Figure S1).

Human cardiac myocytes cells were seeded onto a 12‐well plate covered with slips and then transfected with WT or variant plasmid DNA. Cells were harvested 24 h after transfection. Cells were incubated with rabbit anti‐P27 antibody (dilution 1 : 800; Cell Signaling Technology) diluted in NaCl/P_i containing 5% goat serum (Beyotime) and 0.2% Triton X‐100 at 4 °C overnight, followed by incubation with Cy3‐conjugated goat anti‐rabbit secondary antibody (dilution 1 : 300; Jackson ImmunoResearch, West Grove, PA, USA). Cell nuclei were stained using 4',6‐diamidino‐2‐phenylindole (Servicebio). Image analysis [21, 22] was condicted using a SP8 microscope (Leica Microsystems, Wetzlar, Germany).