Acquastain

Proteins were separated on 4–15% Mini-Protean TGX Stain-Free gels (Bio-Rad) in a Mini-Protean Tetra Vertical Electrophoresis Cell (Bio-Rad). Non-denaturing (native)-PAGE was run in native running buffer (25 mm Tris, 0.19 m glycine, pH 8.3). For SDS-PAGE separation, the native running buffer was supplemented with 1% SDS. PAGE-separated proteins were visualized by staining with AcquaStain (Bulldog Bio). Precision Plus Protein Unstained Protein Marker (Bio-Rad) or High Molecular Weight Native Marker (GE Healthcare) were included for size comparisons.

Noncovalently bound cell surface proteins, including S-layer proteins and S-layer-associated proteins (SLAPs), were extracted from NCK1909 and ΔacmB L. acidophilus NCFM strains using LiCl denaturing salt, as described previously (25 (link)). Proteins were quantified via a bicinchoninic acid assay kit (Thermo Scientific) and visualized via SDS-PAGE using precast 4 to 20% Precise Tris-HEPES protein gels (Thermo Scientific). The gels were stained using AcquaStain (Bulldog Bio) according to the instructions of the manufacturer.

The complexes used in this study that contain either consensus Vif (Vif_con) or LAI Vif (Vif_LAI) (i.e., A3G-Vif_con-CRL5-CBFβ, Vif_con-CBC-Cul5_NTD, Vif_con-CRL5-CBFβ, and A3G-Vif_LAICBC) were purified as previously described in (49 (link), 50 (link), 51 , 52 (link)). Reconstituted complexes were diluted to ~5 μM (different preparations at 1–1.2 mg/ml) in 20 mM HEPES pH 7.5, 300 mM NaCl, 10% Glycerol. Samples were reacted with increasing molar ratios of DSSO (supplemental Table S1) and cross-linking reactions carried out at 37 °C for 30 min at 1000 RPM on an Eppendorf Thermomixer C. All reactions were quenched with 100 mM NH₄HCO₃ or 100 mM Tris pH 8.0, then mixed with SDS-PAGE loading dye. Cross-linked samples were analyzed by SDS-PAGE and stained with MS safe blue stain (AcquaStain, Bulldog Bio). Bands corresponding to cross-linked proteins (as compared with non-cross-linked control samples) were excised and subjected to in gel digest with either trypsin or chymotrypsin (supplemental Fig. S2). Cross-linked peptides were analyzed by LC-MS³ and identified through database searching as described below.

Non-covalently bound cell surface proteins, including S-layer proteins (SLPs) and S-layer associated proteins (SLAPs) were extracted from the Lactobacillus strains using LiCl denaturing salt, as described previously (Johnson et al., 2013 (link)). SLP and SLAP pellets were resuspended in 10% (w/v) SDS (Fisher, Waltham, MA, USA). Proteins were quantified via bicinchoninic acid assay kit (Thermo Scientific, Waltham, MA, USA) and 10 ng was loaded and visualized via SDS-PAGE using precast 4–20% Precise Tris-HEPES protein gels (Thermo Scientific, Waltham, MA, USA). Gels were stained using AcquaStain (Bulldog Bio, Portsmouth, NH, USA) according to the instructions from the manufacturer.
Surface layer associated proteins extracted from the various L. acidophilus strains were identified using LC–MS/MS from the Genome Center Proteomics Core at the University of California, Davis, as described previously (Johnson et al., 2013 (link)). For all analyses, total spectral counts were utilized as a semi-quantitative indicator of protein abundance (Liu et al., 2004 (link)). Two-way clustering of total spectral counts was performed using JMP Genomics (version 5, SAS). Protein domains were identified for analysis using the Pfam protein family database (Finn et al., 2014 (link)).