Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g

Immunoblot analysis was done as described⁵⁵ . Rabbit polyclonal α-MglA^{27 (link)} (dilution 1:5000), α-MglB^{27 (link)} (dilution 1:5000), α-RomR^{32 (link)} (1:5000), α-PilC^{58 (link)} (dilution 1:5000), α-PilO^{59 (link)} (dilution 1:2000), α-PilT^{60 (link)} (dilution 1:2000), α-mCherry (Biovision, dilution 1:15000) and α-MglC antibodies (dilution 1:5000) were used together with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Sigma) as a secondary antibody (dilution 1:10000). Mouse α-GFP (Sigma, dilution 1:2000) and α-EF-Tu (HycultBiotech, dilution 1:5000) antibodies were used together with horseradish peroxidase conjugated sheep anti-mouse immunoglobulin G (GE Healthcare) as a secondary antibody (dilution 1:2000). To generate rabbit polyclonal α-MglC antibodies, His₆-MglC was purified as described (see below) and used for immunization as described⁵⁵ . Blots were developed using Luminata Crescendo Western HRP substrate (Millipore) and visualized using a LAS-4000 luminescent image analyzer (Fujifilm). Proteins were separated by SDS-PAGE as described⁵⁵ .