Ab222772

Following transfection, cells and tissues were lysed for 48 h using RIPA Lysis and Extraction Buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and the protein concentration was measured using Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Following heating at 100°C for 10 min in the presence of a loading buffer, equal amounts of protein lysates (50 µg) were separated using 10% SDS-PAGE (Bio-West Inc., Logan, UT, USA) at 100 V for 1 h, and transferred onto Invitrogen nitrocellulose membranes (Thermo Fisher Scientific, Inc.) at 120 V for 1 h. Following blocking with 5% skimmed milk (diluted with PBS), the membranes were incubated overnight at 4°C with the following primary antibodies: CYP27A1 (ab126785; 1:500; Abcam, Cambridge, MA, USA), c-myc (ab32072; 1:500; Abcam), RB (ab181616; 1:500; Abcam), Ki-67 (ab15580; 1:500; Abcam), CDK2 (ab32147; 1:500; Abcam), p21 (ab109520; 1:500; Abcam), p53 (ab32049; 1:500; Abcam), PDCD4 (ab51495; 1:500; Abcam), SOX2 (ab92494; 1:500; Abcam), β-actin (ab8227; 1:500; Abcam). Subsequently, the membranes were incubated with secondary goat monoclonal (RMG01) to rabbit IgG Fab region (Biotinylated; ab222772; 1:10,000; Abcam) at room temperature for 2 h, and proteins were detected using enhanced chemiluminescence (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).

Immunofluorescence was performed as described previously (33 (link)). HEC-1A cells were plated on glass coverslips in six-well plates and transfected with pre-miR-215 and control miR (mock). At 48 h after transfection, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, and then blocked with goat serum blocking solution (10%; 50197Z; Thermo Fisher Scientific, Inc.) for 20 min at room temperature. Coverslips were stained with the following primary antibodies: Anti-LEFTY2 (1:500; ab229668, Abcam) and anti-E-cadherin (1:500; ab15148, Abcam). Following three washes with NaCl/Pi, cells were incubated with anti-Rabbit antibody (Biotin; 1:10,000; ab222772; Abcam) for 30 min at 37°C. DAPI staining (blue) was used to indicate nuclei at room temperature for 30 min. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). Fluorescence intensities were measured in 300 cells/coverslip and analyzed using ImageJ 1.37 software (rsb.info.nih.gov/ij/index.html; National Institutes of Health, Bethesda, MD, USA).