Af7011

Total protein was extracted from the proximal colon tissues with RIPA buffer containing PMSF and determined by a BCA kit (Keygen Biotech, KGP902, China). The protein samples (60 μg/lane) were loaded on SDS-PAGE gels (Beyotim, P0012A, China) and transferred onto PVDF membranes (Merck, IPVH00010, United States) after electrophoresis. Membranes were blocked with 5% skimmed milk for 2 h and then incubated overnight at 4°C with CACNA1C (1:500, Affinity, DF2267), CACNA1D (1:500, Affinity, DF2267), cyclic adenosine monophosphate (cAMP, 1:10000, Abcam, ab76238), protein kinase A (PKA, 1:500, Affinity, AF7746), and β-Tubulin (1:5000, Affinity, AF7011) antibodies. Afterward, an HRP-conjugated antibody (1:5000, Affinity, S0001, China) were incubated with the membrane. The chemiluminescence (ECL) kit was employed for the membrane visualization by a Tanon 5200 system (Shanghai, China). ImageJ 7.0 was used for quantification.

Intestine and hypothalamus  protein was extracted using RIPA lysis buffer (G2002; Servicebio) and PMSF (G2008; Beyotime). The protein amount was measured using a BCA protein assay reagent (P0012; Beyotime). Equal amounts of proteins were denatured in SDS-PAGE sample loading buffer (P0015L; Beyotime) at 37 °C for 10 min and then separated on a 10% SDS-PAGE gel and transferred onto a PVDF membrane (IPVH00010; Merck Millipore Ltd). Membranes were soaked with 5% nonfat milk in TBST (20 mM Tris·Cl, 150 mM NaCl, and 0.05% Tween-20, pH 7.4) for 1 h at room temperature. Subsequently, membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with Alexa Fluor 790 goat anti-rabbit IgG H&L (1:10000; ab186697; Abcam, Cambridge, UK). Finally, fluorescent signals were collected using an Odyssey infrared imaging system (LI-COR, Lincoln, NE). The intensities of protein bands were quantified with the Image-J 1.53e software (National Institutes of Health, USA), and the values were normalized to β-tubulin (1:2000, AF7018, Affinity) or β-actin (1:2000, AF7011, Affinity). GPR35 polyclonal antibody (YT2015; 1:1000) and CALCR polyclonal antibody (YN2509; 1:1000) were purchased from ImmunoWay Biotechnology Company, and GPR120 antibody (CSB-PA002756; 1:500) and GPR151 antibody (CSB-PA007164; 1:500) were purchased from CUSABIO technology.