Rabbit phospho erk 1 2

Protein lysates of neonatal rat ventricular cells were prepared and subjected to SDS-electrophoresis, as previously described [27 (link)]. Lysates containing 30 µg of proteins were subjected to SDS-polyacrylamide gel (8%) electrophoresis and transferred to a nitrocellulose paper. Antibodies used were rabbit anti-Ca_Vα2δ1 (1:1000, Alomone ACC-015), rabbit Ca_Vα1C (1:1000, Alomone ACC-003), rabbit Ca_Vβ2 (1:5000, Alomone ACC-105), rabbit Ca_Vβ3 (1:500, Alomone ACC-008), rabbit Phospho-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA, 9101S), Total ERK 1/2 (Cell Signaling Technology, 137F5) and rabbit GAPDH (Jackson, West Grove, PA, USA, 111-035-144). Membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with secondary anti-rabbit antibody (1:10,000, Jackson) for 2 h at room temperature. Signal was detected using enhanced chemiluminescence (ECL) substrate.

The cells were washed twice with PBS, and lysed using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, USA) with protease (Thermo Fisher Scientific, USA) and phosphatase (Nacalai Tesque, Japan) inhibitors. For western blot analysis, proteins were separated by SDS-PAGE and transferred onto PolyVinylidene DiFluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C using the following primary antibodies: mouse V5 (Nacalai Tesque, USA), rat FLAG (Novus Biologicals, USA), rabbit ERK1/2, rabbit phospho-ERK1/2, mouse α-Tubulin (Cell Signaling Technology, USA), or mouse α smooth muscle actin (SMA) (Sigma-Aldrich, USA). Bands were detected using Chemi-Lumi One Super (Nacalai Tesque, Japan) and Chemiluminescence detection system LAS-4000 Mini (GE Healthcare, USA).