Ultra high sensitivity chemiluminescence imaging system

BMSCs (3×10⁵ cells seeded in 6-well plates) were lysed in RIPA buffer (Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors. Total protein was quantified using BCA Protein Assay kit (Bio-Rad Laboratories, Inc.). Then, 30 μg/lane protein were loaded onto SDS-PAGE in 5-15% Bis-Tris precast gel to separate the different proteins. Subsequently, samples were transferred to a PVDF membrane (MilliporeSigma) and blocked with 5% non-fat milk for 1 h at room temperature. Then, PVDF membranes were incubated with primary antibodies [H3K9me3, Hp1α, LAP2, RIG-I, phosphorylated TANK-binding kinase 1 (p-TBK1), TBK1 or GAPDH] at 4°C overnight followed by secondary antibodies (1:10,000; HRP AffiniPure Goat Anti-Rabbit, cat. no. EM35111-01 or HRP AffiniPure Goat Anti-Mouse, cat. No. EM35110-01; Beijing Emarbio Science & Technology Co., LTD.) for 1 h at room temperature. The signals were detected using the ECL Immobilon Western Chemilum HRP Substrate (cat. no. WBKLS0500; Merck Millipore) and an ultra-high sensitivity chemiluminescence imaging system (Bio-Rad Laboratories, Inc.) and then quantified using ImageJ 1.48v software (National Institutes of Health).
The antibodies were as follows: H3K9me3 (1:1,000; cat. no ab8898; Abcam), Hp1α (1:1,000; cat. no. 2616; Cell Signaling Technology, Inc.), LAP2 (1:500; cat. no. 611000; BD Biosciences), RIG-I (1:1,000; cat. no. A13407; ABclonal Biotech Co., Ltd.), p-TBK1 (1:250; cat. no. AP1418; ABclonal Biotech Co., Ltd.), TBK1 (1:1,000; cat. no. A3458; ABclonal Biotech Co., Ltd.) and GAPDH (1:3,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.).