Anti crabp2

Immunocytochemical staining was conducted on cell-bearing coverslips by the method described elsewhere [15 (link)]. The antibodies used were: Cyclin D1 (Bioss Inc., Beijing, China; 1:100), Tg (Bioss. Inc., Beijing, China; 1:200), E-cadherin (Bioss. Inc., Beijing, China; 1:100), RAR-β (Bioss. Inc., Beijing, China; 1:150), and PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200). Cells incubated in 0.2% DMSO-containing medium were used as a control. The color reaction was performed by using 3,3′-diaminobenzidine tetrahydrochloride (DAB) after the binding of the primary antibody (Vector Laboratories, Burlingame, CA, USA). For double immunofluorescent staining (IF), mouse anti-CRABP2 and rabbit anti-FABP5 were employed in the working concentrations of 1:120. Briefly, the cell-bearing coverslips were washed with phosphate-buffered solution (PBS, pH 7.4), incubated in 3% H₂O₂ for 10 min and then with anti-CRABP2 (1:120; Proteintech, Chicago, IL, USA) and anti-FABP5 (1:120; Proteintech, Chicago, IL, USA) at 4 °C for one night in a humid chamber. Finally, the coverslips were co-incubated with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 60 min in the dark, sealed with fluorescence mounting medium, and observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan).

Proteins were extracted from the drug-treated cells under different experimental conditions. Equivalent amounts of sample proteins were separated by 8–12% SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% non-fat dry milk (Sigma-Aldrich) in TBS-T (10 mM Tris-HCl, PH8.0, 150 mM NaCl, and 0.5% Tween 20) for 2 h at room temperature and incubated with anti-caspases-3 (Abcam Inc., Cambridge, UK;1:500), anti-CRABP2 (Proteintech, Chicago, IL, USA; 1:200), anti-DNMT1 (Bioss. Inc., Beijing, China; 1:300), anti-DNMT3A (Bioss. Inc., Beijing, China; 1:1,000), and anti-DNMT3B (Bioss. Inc., Beijing, China; 1:300) primary antibodies overnight at 4°C. After three washes with TBS-T, the membrane was incubated for 1.5 h with HRP-conjugated anti-mouse or anti-rabbit IgG (Zymed Lab, Inc.). Protein expression was detected using chemiluminescent ECL reagents (Roche GmbH, Mannheim, Germany). A Gel-Pro analyzer was used to measure the density of bands. Simultaneously, the expression of a target protein was normalized to that of β-Actin.

RIPA Lysis Buffer (CWBIO, Beijing, China) was used to extract the proteins in cells treated with dezocine for 48 h. Following which the concentration was determined by BCA kit (CWBIO), the protein samples (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF members (Millipore, Billerica, MA, USA). After being blocked with 5% dried skimmed milk for 1 h, the members were incubated with primary antibodies at 4°C overnight, and then incubated with HRP-conjugated secondary antibodies at room temperature. An enhanced chemiluminescence reagent (CWBIO) was performed to visualize the blot bands. The antibodies, including anti-Bcl-2 (Cat no. 12789-1-AP), anti-Bax (Cat no. 50599-2-Ig), anti-p-Akt (Cat no. 66444-1-Ig), anti-mTOR (Cat no. 20657-1-AP), anti-GAPDH (Cat no. 10494-1-AP), and anti-CRABP2 (Cat no. 10225-1-AP) were obtained from Proteintech Group (IL, USA); anti-cleaved Caspase 3 (Cat no. 9661), anti-Akt (Cat no. 9272), anti-p-mTOR (Cat no. 5536), anti-p70s6k (Cat no. 9204), and secondary antibodies were obtained from Cell Signaling Technology (Danvers, USA).