Laemilli buffer

ARVM protein was isolated using RIPA buffer as previously described [26 (link)]. Samples were prepared in Laemilli Buffer (Bio-Rad) with 2.5% βME, separated using 15% Criterion gels (Bio-Rad) as described above. PLNpSer16 (Badrilla) was incubated first at 4°C overnight followed by secondary goat anti-rabbit (Jackson Immunology Research Laboratories). The membranes were then incubated with calsequestrin (Thermo Fisher) overnight at 4°C followed by secondary goat anti-rabbit (Jackson Immunology Research Laboratories). Lastly the membranes were incubated with an antibody against PLN (Thermo Fisher) overnight at 4°C and secondary sheep anti-mouse (Jackson Immunology Research Laboratories). PLNpSer16 and total PLN proteins were normalized to calsequestrin.

Nuclear protein extracts were prepared in Laemilli Buffer (Bio-Rad) with 2.5% β-mercaptoethanol (βME), boiled for 5 minutes at 95°C, separated using 10% Criterion gels (Bio-Rad) at 140V for 2h and transferred onto 0.2μM nitrocellulose membranes (GE Healthcare Life Sciences) at 80V for 80 min. Membranes were blocked with 10% milk in Phosphate-buffered saline (PBS) plus 0.1% Tween. Primary antibody SRF (Santa Cruz) was incubated at 4°C overnight, and secondary goat anti-rabbit (Jackson Immunology Research Laboratories), was applied for 1h. Proteins were normalized to Lamin A/C-HRP conjugated (Santa Cruz).

Samples were prepared in Laemilli Buffer (Bio-Rad) with 2.5% β-mercaptoethanol (βME), boiled for 5 minutes at 95°C as described for SRF. Primary antibodies were incubated overnight, and secondary antibodies were incubated for 1 hour at room temperature as described above. Calnexin antibody was purchased from Abcam, Catalytic active subunit of PKA and PP2Cε antibodies were purchased from R&D systems.
All Western blots were incubated with enhanced chemiluminescence reagents (West Pico or West Femto, ThermoFisher Scientific) and exposed to autoradiography film. Protein abundance was quantified using ImageJ (Version 1.49V).