Anti cav1

Immunoprecipitation was performed using a Protein G Immunoprecipitation Kit (Sigma-Aldrich)^{6 (link)}. Cell pellet was resuspended in 1.0 mL of lysis buffer (20 mM sodium phosphate, 150 mM sodium chloride, 10% glycerol, 1 mM ethylenediaminetetraacetic acid, 0.5% Triton-X 100 [pH 7.2]) and complete TM protease inhibitor cocktail (Roche, Basel, Switzerland) at ∼ 1 mg/mL. The suspension was placed on ice for 1 h and centrifuged at 10,000×g at 4 °C for 15 min. The cleared lysate was incubated at 4 °C for 1 h with 2 μg of a monoclonal anti-flag antibody (Sigma-Aldrich) against flag-tagged Ca_Vβ2 protein. Protein G Sepharose (50 μL) was added to the samples, which were incubated for 16 h at 4 °C. The immunoprecipitate was washed five times with 1 mL of immunoprecipitation buffer before being eluted with 60 μL of Laemmli buffer. The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca_V1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca_V1.2. Anti-rabbit IgG alkaline phosphatase conjugate (Promega) was used for immunodetection.

Primary antibodies used were: Rabbit polyclonal anti-Cav1.2 (Alomone Labs), Phospho Serine-1928 (Badrilla) and Rabbit polyclonal anti-S-nitrocysteine (Abcam), Mouse monoclonal sarcomeric α-actinin (Sigma). Secondary antibodies used were: Donkey anti-mouse Dylight 549, Donkey anti-rabbit Dylight 488 (Jackson ImmunoResearch), rabbit GAPDH antibody (Sigma-Aldrich).

HL-1 cells were lysed using RIPA buffer (Thermo Fisher, Waltham, MA) containing protease inhibitor cocktail (Roche, South San Francisco, CA). Equal amounts of protein (10–20 μg) were loaded into wells of 7% or 3–8% tris-acetate gels, subjected to SDS-PAGE, and transferred to a 0.45 μm PVDF membrane (Thermo Fisher). Western blot analyses were performed using the following primary antibodies: anti-Flag (Sigma-Aldrich), anti-Kv4.3 (Alomone, Jerusalem, Israel), anti-Kv11.1 (Alomone), anti-Nav1.5 (Alomone), anti-Cav1.2 (Alomone), and anti-GAPDH (Millipore, Billerica, MA). The immunodensity of bands corresponding to targeted proteins were quantified using NIH ImageJ software, and values were corrected for protein loading by dividing them by GAPDH immunodensity signals in the same lanes. Subsequently, corrected values obtained from Western blots loaded with protein lysate from BKα-transfected cells were normalized to corrected values obtained from Null-transfected cells corresponding to the same ion channel. According to this analysis, by definition the average normalized intensity of Null transfection is unity. For Western blots designed to detect Kv11.1, both bands of the Kv11.1 doublet revealed by anti-Kv11.1 blotting were included in calculations.

Cells were washed with PBS twice and lysed in RIPA lysis buffer (Beyotime) at 4°C for 2 hr. Samples were separated using 10% Tris‐glycine SDS‐polyacrylamide gel (Invitrogen) and transferred to PVDF membranes with a current of 200 mA for 2 hr. After blocked with 5% albumin from bovine serum (BSA) in PBST (PBS with 0.1% Tween), membranes were incubated overnight at 4°C with the following primary antibodies: anti‐GAPDH (CWBIO, CW0100), anti‐ALP (Abcam, ab108337), anti‐Runx2 (Cell Signaling Technology, #12556), anti‐OCN (Santa Cruz Biotechnology, sc‐390877), anti‐GSK3β (Cell Signaling Technology, #9832), anti‐β‐Catenin (Cell Signaling Technology, #8480), anti‐phospho‐GSK3β (Cell Signaling Technology, #9323), anti‐active‐β‐Catenin (Millipore, 05‐665), and anti‐Ca_v1.2 (Alomone labs, Acc‐003). Then, the protein bands were incubated with secondary antibody (Jackson) and visualized using an enhanced chemiluminescence kit (Pierce) according to the manufacturer's instructions. The quantitative data of Western blot were analyzed by Image J (National Institutes of Health).

The total proteins of the cells were lysed in a RIPA buffer containing 1% protease inhibitors (Beyotime, Shanghai, China) and collected. After SDS-PAGE, the proteins were transferred onto PVDF membranes. The primary antibodies were anti-Cav1.2 (Alomone, Jerusalem, Israel), anti-Nav1.5 (Alomone, Jerusalem, Israel), anti-Kv4.2 (Abcam, Cambridge, England), anti-HOXa10 (ABclonal, Wuhan, China), and anti-β-Actin (ABclonal, Wuhan, China). The HRP-conjugated secondary antibody HRP-labeled goat anti-rabbit IgG (Absin, Shanghai, China) was used.