Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with TgROP18

He, Cheng; Kong, Ling; Zhou, Lijuan; Xia, Jing; Wei, Haixia; Liu, Min; Peng, Hongjuan

HFF cells infected with T. gondii RH-ROP18-eGFP-FLAG for 36h and uninfected controls were lysed and clarified by centrifugation as described above. For co-immunoprecipitation (Co-IP), mouse monoclonal vimentin or FLAG antibodies were added to the clarified cell extracts and incubated at 4°C for 1 h. Then Protein A-Agarose (Santa Cruz) was added to the mix and incubated for 1h at 4°C with rotation. Beads were collected by centrifugation at 1,000g for 5min at 4°C, and then washed for four times with PBS with rotation for 10 min at 4°C. Finally, beads were collected by centrifugation as described above, resuspended in SDS-PAGE sample buffer, and then boiled for 10 min. Boiled samples were centrifuged briefly to eliminate debris, loaded for SDS-PAGE, and then analyzed with rabbit monoclonal anti-vimentin or polyclonal anti-DDDDK (for FLAG tag detection) antibodies by WB (Supplementary Material).