Hsp60

HeLa cells were infected with C. trachomatis L2-wild type and L2-ΔCT228; at different time points during infection, cells were lysed with 2X Laemmli Sample Buffer (Biorad, Hercules, CA). Protein samples were separated by SDS-PAGE electrophoresis (12 or 15% gel) and transferred to 0.2 um nitrocellulose membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk/5% BSA in 1X Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies diluted in 5% non-fat milk/5% BSA in 1X TBST at 4°C overnight followed by incubation with horseradish peroxidase conjugated anti-rabbit/mouse antibodies (Cell Signaling, Danvers, MA) for 1 h at room temperature. Detection was performed by enhanced chemiluminescence using SignalFire ECL reagents (Cell Signaling Technology, Danvers, MA). Immunoblot images were acquired using the Fluorchem E FE0622 system (ProteinSimple, San Jose, CA). Primary antibodies to MLC2 (Millipore Sigma, Burlington, MA), p-MLC2 (S19) (ThermoFisher Scientific), HsP60 (ThermoFisher Scientific), GAPDH (Santa Cruz), and MYTP1 (Cell Signaling, Danvers, MA) were used herein.