Goat anti rabbit antibodies

Daoy was cultured in DMEM medium containing 10% serum for 24 h, then added imipramine blue 0.25 µM for 48 h. Whole cell lysates were harvested in lysis buffer (Cell Signaling Technology, Danvers, MA) for Western Blot. Western blot was performed with the following primary antibodies: PAK1, Catalase, ERK1/2, Cyclin D2 and Gapdh (Cell Signaling Technology, Danvers, MA); BCL2, Cleaved -PARP and Caspase-3(ABCAM, Cambridge, MA, USA). Goat anti-rabbit antibodies (Santa Cruz, Dallas, TX, USA) were used and the immunoreactive bands were detected by ECL. Original images can be found at Figure S1.

For the examination of PilA expression, whole cell lysates from 5 × 10⁷ cells were separated by 15% SDS-PAGE and analyzed by immunoblotting with anti-PilA antibodies38 (link) as the primary antibody and goat-anti-rabbit antibodies conjugated to alkaline phosphatase as the secondary antibody (Santa Cruz Biotechnology). To examine strains for the assembly of T4P, pili isolated from 5 × 10⁸ cells were prepared by shearing and a differential precipitation as previously described12 (link)39 (link). Briefly, surface pili were sheared from cell suspensions by vortexing for 3 minutes. Intact cells were removed by two rounds of centrifugation at 16,000×g for 10 minutes. MgCl₂ was added to the supernatant at a final concentration of 100 mM. After incubation on ice for 60 min, the pilus fraction was collected by centrifugation at 16,000 g for 15 min and resuspended in SDS-PAGE loading buffer prior to SDS-PAGE and immunoblotting analysis with anti-PilA antibodies.