Nmhc 2a

Immunoblotting was performed as described by Zhang et al. (2012) , with the exception that proteins were transferred to nitrocellulose membranes (Invitrogen). Signal was detected with fluorescence-linked secondary antibodies on a Li-Cor Odyssey infrared imager. Primary antibodies were NMHC 2A (1:50,000; BioLegend), NMHC 2C (1:5000; BioLegend), GFP (1:5000; Abcam), p-paxillin (1:1000; BD Transduction Laboratories), paxillin (1:20,000; BD Transduction Laboratories), and GAPDH (1:5000; Meridian).

Mouse embryos were collected in phosphate-buffered saline (PBS) and directly immersed in 4% paraformaldehyde (PFA) in PBS (pH 7.4) at 4°C overnight. Following fixation, the samples were washed with PBS and stored in PBS at 4°C for further analyses. Back skins and hearts were dissected from fixed embryos and then stained by wholemount immunofluorescence staining as previously described (Yamazaki et al., 2017 (link)). The following primary antibodies were used in this study: polyclonal antibodies against NMHC 2A (1:1,000, Biolegend), NMHC2B (1:3,000, Biolegend), p-paxillin (1:1,000; BD Transduction Laboratories); monoclonal antibodies against PECAM1 (CD31, 1:200, BD Pharmingen), GFP (1:200, ab183734, Abcam). Fluorescence secondary antibodies used were Alexa 488 goat anti-rabbit IgG or Alexa 594 goat anti-mouse IgG (1:250, Invitrogen, Carlsbad, CA). Confocal microscopy was carried out either on a Leica TCS SP5 or on a Zeiss LSM 880. In all cases, when possible, comparison was made among littermates. For each genotype we analyzed at least three to five mice. We found no abnormalities in NM2A and NM2B single heterozygous mice or NM2A/2B double heterozygous mice compared with the littermates that expressed no Cre. Littermates containing no cre recombinase were used as controls for conditional ablated mice.