Cacna1c

Protein extract preparation from tissue samples and western blotting were performed as described previously [21 (link)]. Primary antibodies against the following antigens were used: CACNA1C (guinea pig, 1:1000, Alomone Labs, Jerusalem, Israel), SRF (rabbit, 1:500, Santa Cruz Biotechnology, Dallas, TX), or GAPDH (rabbit, 1:1500, Cell Signaling Technology, Danvers, MA). All antibodies were incubated in 5% Blotto, non-fat dry milk (Santa Cruz Biotechnology, Dallas, TX). HRP-conjugated goat anti-guinea pig (AP108P), goat-anti-rabbit (AP307P), or goat anti-mouse (AP308P) secondary antibodies (1:50,000; EMD Millipore, Billerica, MA) were used with an enhanced chemiluminescence method (Amersham ECL Advantage, GE Healthcare Life Sciences, Pittsburgh, PA). The protein bands were captured using a CCD-camera system (EC3 410 Imaging System; UVP, Upland, CA) and analyzed with VisionWorksLS software (Version 6.8; UVP, Upland, CA). The expression level of CACNA1C and SRF was normalized to the ratio of CACNA1C and SRF area density divided by GAPDH area density. For quantitative measurements, the values of relative expression for CACNA1C and SRF in KO5D, KO10D, and KO15D small intestine were compared to the expression in WT10D small intestine (set to 1.0).

Blebbistatin and di-4-ANEPPS were obtained from Cedarlane (Burlington, Ontario). Troponin-I was obtained from Abcam (Cambridge, UK). CACNA1C, CX43, IL1β, IL6, KCNQ1, NLRP3, PLN, RYR2, SCN5A, TGFβ1, and GAPDH qPCR probes as well as DAPI and WGA were obtained from Invitrogen (Waltham, MA, USA).
Primary antibodies for immunoblot experiments included: BIN1 (Novus Biologicals, NBP1-89102), CACNA1C (Alomone labs ACC-003), CAV (Novus Biologicals, NBP3-16503), IL6 (ThermoFisher ARC0962), JPH2 (ThermoFisher, PA5-141187), IL1β (ThermoFisher PA5-46956), KCNQ1 (NeuroMab clone N374/10), NLRP3 (Novus Biologicals NBP1-77080SS), SCN5A (Alomone labs ASC-005), RYR2 (Thermofisher MA3-916), SERCA2a (Thermofisher MA3-910).