St ht31

mpkCCD_cl4 cells (gift from Alain Vandewalle, Paris) were cultured as previously described^{25 (link)}. The mpkCCD cells were seeded on semipermeable filters (Transwell, 0.4-µm pore size; Corning Costar). For immunofluorescence and western blot analysis, 0.33-cm² and 4.67-cm² filters were used, respectively. The mpkCCD cells were cultured for 5 days, with changes of the medium daily. St-Ht31 (Promega Corporation, Madison, WI, USA), St-Ht31P (Promega Corporation), FMP-API-1 (Sigma–Aldrich Corporation, St. Louis, MO, USA), FMP-API-1/27, FMP-API-1/28, dDAVP (Sigma–Aldrich Corporation), and H89 (Sigma–Aldrich Corporation) were applied to the basolateral side of the mpkCCD cells.

φ450-cAMP, φ575-cAMP, φ575-OMe-cAMP and φ450-free dye were either purchased or obtained as a gift from Biolog Life Science Institute (Bremen, Germany); MitoTracker Red was purchased from Life Technologies (Carlsbad, CA); EGFP from Biovision Inc. (Milpitas, CA), ST-Ht31 and ST-Ht31P from Promega (Madison, WI). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

In-house rabbit polyclonal CatSper1 (Ren et al., 2001 (link)), CatSper3 (Qi et al., 2007 (link)), CatSperε (Chung et al., 2017 (link)) antibodies were described previously. Polyclonal CA-IV antibody (M-50) was purchased from Santacruz. Monoclonal antibodies were purchased from BD Biosciences: anti-caveolin1 (clone 2297); EMD Milipore: anti-phosphotyrosine (clon4G10), anti-acetylated tubulin (clone 6-11B-1), anti-HA agarose (clone HA-7); Thermo Scientific: anti-HA magnetic beads; and Cell Signaling Technology: β-actin (clone 13E5) and HA (clone C29F4). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were from Jackson Immunoresearch. PNA-Alexa 568, WGA-Alexa 555, WGA-Alexa 647, goat anti-mouse IgG (Alexa 488 or 647), and goat anti-rabbit IgG (Alexa 568 or Alexa 647) were from Invitrogen. H89, calyculin A, and calpain inhibitor I were purchased from Calbiochem. ST-Ht31 was from Promega. Calpain inhibitor II and III were from Enzo life science. All other chemicals were from Sigma-Aldrich unless indicated.