Anti rabbit sc 2004

PLC/PRF/5 and Huh7 cells were collected, washed twice with pre-cooled PBS, and then lysed for 30 min on ice using a cell lysis buffer (Beyotime, P0013, Haimen, China), containing 1 percent protease inhibitor (PMSF), and 1 percent phosphokinase inhibitor. The supernatant protein lysate was then collected. Protein concentration was measured by a Pierce BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA). A PVDF membrane was then used to transfer the separated cellular lysates that had been separated using a 10% SDS-PAGE gel. The chosen primary antibodies were probed with the PVDF membrane overnight at 4 °C after it had been masked with 5% BSA buffer for an hour at room temperature. The primary antibodies against COPZ1 (1:1000; Proteintech, 20440-1-AP, Sankt Leon-Rot, Germany) and GAPDH (1:5000; AM102013) were used. After that, 1×TBST (washing buffer) washed the mem-brane 3 times for 10 min each time. After being incubated with a secondary antibody for an hour at room temperature, the protein membrane was washed with 1×TBST three times for a total of ten minutes. The secondary anti-mouse (sc-2005) and anti-rabbit (sc-2004) antibodies were purchased from Santa Cruz Biotechnology. An ECL chemiluminescence hypersensitive colorimetric kit and a chemiluminescent imaging system were used for signal generation and detection.

Western blot analysis was performed in LOVO and SW480 cells using the following primary antibodies: GLUT1 (21829-1-AP, Proteintech) and α-tubulin (T 6074, Sigma-Aldrich). Briefly, the cell pellet was mixed with LDS sample buffer (Life Technologies) and sample reducing agent (Life Technologies), according to the manufacturer’s protocol. Proteins extracted were loaded onto a SDS-PAGE (Novex 4–12% Bis–Tris gel, Life Technologies), transferred to a nitrocellulose membrane by iBlot (Life Technologies), and blocked 1 h with 1× TBST buffer (Fisher Scientific) with 5% w/v nonfat dry milk. The membranes were incubated in blocking buffer at 4 °C overnight with primary antibody against GLUT1 (1:1000) or 1 h at room temperature for α-tubulin (1:3000). Subsequently, the membranes were washed three times with Tris-buffered saline with Tween-20 (TBST) and incubated 1 h with secondary antibodies (1:5000; anti-rabbit sc-2004, Santa Cruz Biotechnology; or 1:3000; anti-mouse A9044, Sigma-Aldrich). The signal was obtained using the Novex ECL Chemiluminescent Substrate Reagent kit (WP 20005, Invitrogen) and the images were developed and quantified using the Chemidoc System (Bio-Rad).