To identify OCEL1-binding proteins in Escherichia coli, we set two experimental groups (A and B). Groups A and B represented the expression of GST protein alone or GST-OCEL1-P81A fusion protein in bacteria respectively. The cultures of E. coli BL21 transformed with pGEX-4T1 or pGEX-4T1-OCEL1-P81A were induced with 0.3 mM isopropyl-β-D-thiolgalactopyranoside (IPTG) at 18°C for 6 h. The harvested bacteria were suspended in PBS containing 1% Triton X-100 (PBST) and protease inhibitors and then lysed by sonication. GST resins were added to the bacterial lysates and mixed overnight at 4°C. After 4 washes with PBS, the enriched proteins were were subjected to 10% SDS-PAGE gel. After Coomassie Blue staining, bands corresponding to each group were collected and analyzed by mass spectrometry(LC-MS/MS) in ProteinGene Biotech (Wuhan, Hubei, China). All acquired raw data were processed with pFind software (version 3.1.2) and searched against the NCBI E. coli protein database (https://www.ncbi.nlm.nih. gov/protein). After identifying the associated proteins in each group, we compared them with each other. The proteins enriched in group B compared with those in group A were considered as OCEL1 associated proteins.