Anti alpha 1 sodium potassium atpase

Antibodies used for Western blot analysis and coimmunoprecipitation were anti-HA-POD (clone 3F10, 12158167, Roche), anti-Flag-POD (M2, A8592, Sigma), anti-Flag (M2, F3165, Sigma), anti-Myc (clone 9E10, M5546, Sigma) and anti-Lfng (A-19, sc-8239, Santa Cruz Biotechnology, Inc.), and a rat monoclonal anti DLL3 antibody 4H3 (generated against a peptide comprising amino acids 557-571of mouse DLL3) as described in [37 (link)]. Primary antibodies used for immuncytochemistry were anti-Flag (M2), anti-HA (3F10), anti-GM130 (610823,BD Biosciences), anti-Alpha 1 sodium potassium ATPase (ab7671, Abcam), anti-DLL3 (4H3), anti-DLL1 (H-265, sc-9102, Santa CruZ Biotechnology, Inc.) and anti-DLL1 (1F9, [37 (link)]. Secondary antibodies used were: goat anti-mouse-Alexa633 (A21052, Invitrogen), goat anti-rat-Alexa488 (A11006, Invitrogen), goat anti-rabbit-Alexa488 (A11034, Invitrogen), goat anti-rat-Alexa555 (A21434, Invitrogen).

The primary antibodies used in this study were anti-CACNA1G [20 (link)], and anti-alpha 1 sodium potassium ATPase (Abcam, Cambridge, UK).
HeLa and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nakarai Tesque, Kyoto, Japan) supplemented with 10 % fetal bovine serum and penicillin/streptomycin (PS) in a 37 °C incubator with 5 % CO₂. For immunofluorescence analysis, cells were grown on chamber slides (SCS-008; Matsunami, Osaka, Japan) coated with poly-l-lysine (Sigma-Aldrich, St. Louis, MO, USA), and were transiently transfected with pCMV-SPORT6-CACNA1G using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. After 48–72 h, the cells were fixed in 4 % paraformaldehyde, washed with phosphate-buffered saline (PBS), blocked, and permeabilized with 0.2 % Tween20. Cells were incubated overnight at 4 °C with anti-CACNA1G and anti-alpha 1 sodium potassium ATPase antibodies and then treated with secondary antibodies. Images were obtained using confocal microscopy (LSM510; Carl Zeiss, Jena, Germany). The nuclei were visualized using DAPI.
Cells for whole-cell patch clamping were grown in glass-bottom plates (μ-Dish 35 mm low; ibidi, Martinsried, Germany) for 24 h following transfection with SPORT6-CACNA1G-IG using Lipofectamine LTX.