Sc 5770

The livers and jejunum tissue were homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer [50 mM Tris HCl, 1 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1.0% Nonidet P-40, and phenylmethane sulfonyl fluoride (PMSF), pH 8.0]. The homogenate was centrifuged at 12,000 rpm for 20 min at 4°C. The supernatants were collected for Western blot analysis, which was performed as described previously (Kong et al., 2015 (link)). The antibodies used in the present study included anti-Mrp-2 (1:500; sc-5770, Santa Cruz; California, USA), anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (1:20,000; YM3029, Immunoway; Texas, USA), anti-UGT1A1 (1:4,000; ab194697, Abcam; Cambs, UK), anti-P glycoprotein (1:1,000; ab170904, Abcam; Cambs, UK), anti-CES2 (1:1,000; DF6433, Affinity; Cambs, UK), and anti-Bcrp (1:200; sc-69988, Santa Cruz; California, USA). The gray intensities of the bands were quantified using ImageJ software (National Institute of Health, MD, USA).

The reagents for the fluorescence-based quantitative RT-PCR reactions and the Western-blot reactions and Immunohistochemistry detection were prepared using the manufacturer's instructions. The antibodies from goat for ABCC2 (SC-5770), the antibodies from rabbit for OATP1B2 (SC-134460), donkey anti-goat IgG-HRP, and goat anti-rabbit IgG-HRP were provided by Santa Cruz Biotechnology, Inc. The Rabbit anti-GAPDH (AB-P-R001) antibody was provide by Hangzhou Goodhere Biotechnology Co., Ltd. The following probes and primers were used for mRNA analysis: ABCC2 forward primer: 5′-CTG TCC ATG CTT CCC ATG GT-3′, reverse primer: 5′-GGC GAA TGG CAG ATG TGT CT-3′, probe: 5′-FAM-CTC ATC GAT CCT CCA GGC CAG TGT TT-BHQ1-3′; OATP1B2 forward primer: 5′-CAG TGG CAG GCT TAA CAA CCT-3′, reverse primer: 5′-GGA TCC CAT GTG TTC GTT GAG-3′, probe: 5′-FAM-TGG AGA ACA AGG TCC TTG CTG ACT-BHQ1-3′; and the housekeeping gene GAPDH forward primer: 5′-AGG GCT GCC TTC TCT TGT GA-3′, reverse primer: 5′-AAC TTG CCG TGG GTA GAG TCA-3′, probe: 5′-FAM-CCA TCA ACG ACC CCT TCA TTG ACC TC-BHQ1-3′. The probes were synthesized using an Invitrogen ABI 3900 high-throughput DNA synthesizer.