Anti ha tag

Cells were washed in PBS and fixed in 4% paraformaldehyde for 30 min. After 3 washes in DPBS, cells were permeabilized with 0.3% Triton-X-100 for 60 min, blocked with PBS containing 2% BSA for 60 min and incubated with primary antibodies overnight at 4 °C. Then, the cells were treated with secondary antibody for 60 min at room temperature and mounted with Hoechst 33,342 (Sigma, Beijing, China) after 3 washes in DPBS.
The following antibodies were used in this study: anti-Myc-Tag (Abmart, Shanghai, China, A#M20002), anti-HA-Tag (Abmart, Shanghai, China, #M20003), anti-His-Tag (Abmart, Shanghai, China, #M20001), anti-Flag-Tag (Genscript, Nanjing, China, #A00170) and anti-dCas9 (Genscript, Nanjing, China, #A01885). Goat anti-rabbit secondary antibody (1:5000) (Santa Cruz, Dallas, TX, USA, #sc-2004). Samples were assessed by fluorescence microscopy at 530 nm and 480 nm excitation wavelengths (Olympus BX51, Olympus, Tokyo, Japan).

At 48 h post-transfection, cells were collected for protein isolation using RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor (Invitrogen). A total of 10~25 µg proteins were loaded on SDS-polyacrylamide gel and electrophoresed on 5% concentrated gel at 90 V for 30 min followed by separating gel at 120 V for 90 min. The proteins were then transferred to a polyvinyl difluoride membrane (Millipore, Shanghai, China). After blocking with 5% skimmed milk at 37 °C for 2 h, membranes were incubated at 4 °C overnight with anti-HA tag (1:5000 dilution; Abmart, Shanghai, China), -C/EBPα (1:1000 dilution; Abcam, Shanghai, China), -ACSL1 (1:1000 dilution; ABclonal, Wuhan, China), -GAPDH (1:5000 dilution; ABclonal, Wuhan, China), -β-tubulin (1:1000 dilution; Abcam), or -β-actin (1:2000 dilution; Abmart) primary antibodies. GAPDH, β-tubulin and β-actin were used as control in the experiments. The results were detected on UVP ChemStudio^TM PLUS touch produced by Analytik Jena US (Upland, CA, USA). Gray value analysis was conducted by ImageJ software (version 1.51j8) to calculate relative intensities of the bands.