Western Blot Protein Detection Protocol

Protein extracts were prepared by cell lysis in boiling 1% SDS buffer (18 (link)). Protein extracts (and Co-IP samples) were then mixed with loading buffer and denatured (10 minutes, 95°C–100°C), before separation by SDS-PAGE and transfer to nitrocellulose membranes (Bio-Rad). Membranes were blocked [5% milk or 3% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST)], incubated with primary antibodies (Table S5), washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech or Thermo Fisher Scientific, 1:10,000–1:50,000), and then washed again with TBST. Membranes were developed using HRP substrate [ECL Prime (GE Healthcare) or SuperSignal West Pico PLUS (Thermo Fisher Scientific)]. Membranes were stripped with Restore Plus (Thermo Fisher Scientific) and blocked before the detection of additional targets. Band intensities were quantified using Image Quant TL (GE Healthcare). Expression levels of bacterial and host proteins were normalized to the expression of the Chlamydia protein Slc1 and host β-actin, respectively.