Ab84814

Antibodies against Cav1.2 (ab84814 and ACC‐003) were from Abcam (Cambridge, UK) and Alomone, respectively, which produced identical Western blots. Those against β‐actin (ab6276), MAP‐2 (ab32454), ERα (ab3573), ERβ (ab104804), calcineurin (ab3673), Mdm2 (ab16895), and ubiquitin (ab19247) were purchased from Abcam. pSer1928‐Cav1.2 (A010‐70) was from Badrilla (United Kingdom). 17β‐estradiol (E2758), propylpyrazoletriol (PPT, H6036), diarylprepionitrile (DPN, H5915), E2‐BSA (E5630), ICI 182,780 (V900926), nifedipine (N7630), FK506 (F4679), MG132 (C2211), ammonium chloride (A0171), chloroquine (C6628), carfilzomib (791938), and other reagents were purchased from Sigma (St. Louis, MO, USA). Lipid solvents were made in stock solutions in DMSO (1:1,000 ~ 2,000).

For the CaV1.2 measurements by immunocytochemistry, the cells were seeded on glass cover slips and grown in Petri dishes with complete medium, at a density of 10,000 cells/cover slip. After several days of cultivation, cells were transferred to a new Petri dish, washed 3 times with PBS (Biochrom, Holliston, MA, USA) and fixed with 4% paraformaldehyde (PFA) (Sigma Aldrich, St. Louis, MO, USA) at RT for 20 min. After fixation, the glass slips with the cells were washed twice with PBS and permeabilized with 0.1% Triton X-100 at RT for 20 min. Cells were washed twice with PBS and blocked with 1% bovine serum albumin (BSA) in PBS at +37 °C for 30 min. Antibody against the CaV1.2 subunit (ab84814, Abcam, Cambridge, UK) was prepared in 1% BSA/PBS (1:100) and incubated in 37 °C incubator with 5% CO₂ for 1 h. Secondary anti-mouse Ig-G1-Alexa488 fluorescent antibody (a21200, Thermo Fischer Scientific, Waltham, MA, USA) was added (1:500) and incubated at 37 °C for 30 min. After that, the cells were washed three times with 1% BSA/PBS solution, stained with 1 µM DAPI (4′,6-diamidino-2-phenylindole) dye for 30–60 s (Vector laboratories, Burlingame, CA, USA), washed with water, put on the objective glass and analyzed with fluorescent microscope (EVOS M7000).