C. Analysis of detergent-resistant FadA polymers in wild-type Fn and its mutants by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and Western blot analysis using mAb 7H7. Fn12230 (wt), fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam (lam) were grown to log (OD600 0.3-0.4) or stationary phase (OD600 >0.8), E. coli MC4100, ∆curli, and ∆csgA were grown to stationary phase. The bacteria were harvested by centrifugation. Following sequential incubation in lysis buffer containing 2 mg/ml lysozyme and 1% sarkosyl, the insoluble pellets were obtained by centrifugation. An aliquot of 100 μg of each pellet was loaded onto 1.7% agarose gel followed by overnight electrophoresis in 0.5xTAE and 0.1% SDS. Following transfer to nitrocellulose membrane, Western blot was performed using mAb 7H7 at 1:4,000 dilution to detect the FadA proteins or anti-CsgA antibody at 1:15,000 dilution. The heterogeneously large detergent resistant FadA polymers were detected in wild-type Fn in the stationary phase, defective in US1 and lam, and absent in all in log phase. 