The epithelial cells were infected with bacteria to a MOI of 100. The bacteria were not removed after addition to the epithelial cells. After incubation for the indicated time points the cells were washed twice with PBS and directly placed on ice. The cells were lysed with 50 μl of 1X sample buffer (63 mM Tris-HCl pH 6.8, 25% glycerol, 1% SDS, 5% 2-mercaptoethanol), boiled for 10 min at 95°C and stored at −20°C until use. Thawed samples were centrifuged for 1 min at 10,000 × g and 15 μl was loaded on 10% acrylamide SDS-PAGE gels. After separation, the proteins were transferred to Immobilon-P PVDF membranes (Millipore) using a semi-dry transfer system (Bio-Rad). The membranes were washed in water and blocked for 1 h in 5% skim milk powder (Sigma Aldrich) in PBS at room temperature. The membranes were incubated overnight at 4°C with antibodies against EGR1 (Abcam, ab194357, 1:10,000 dilution) and β-actin (Millipore, MAB1501, 1:2000 dilution) in 1% skim milk powder in PBS. After washing 3 times with PBS, the membranes were incubated with IRDye800-conjugated goat-anti-rabbit and IRDye680-conjugated goat-anti-mouse antibodies (LI-COR, 1:10,000 dilution) for 1 h at room temperature. Bands were visualized using an Odyssey IR scanner (LI-COR).