Alexa fluor 488 and alexa fluor 546 594 coupled secondary antibodies

Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies raised against human S100A11 (1:1000 dilution) (ProteinTech Group), Annexin A1 (1:1000 dilution) (Abcam), Annexin A2 (1:1000 dilution) (BD Transduction Laboratories), heat shock cognate 70 kDa protein (1:6000 dilution) (Hsc70; N69, kindly provided by Boris Margulis, Russian Academy of Sciences, St. Petersburg, Russia), α-tubulin (1:5000) (Abcam) and ErbB2 (1:1000) (Fischer Thermo Scientific, MA) was used. This was followed by appropriate peroxidase-conjugated secondary antibodies (DAKO). Immunocytochemistry: cells on coverslips were fixed in paraformaldehyde and stained with indicated primary antibodies (1:300 dilution) including S100A11 and β-actin (Sigma). Samples were incubated with the appropriate Alexa Fluor-488– and Alexa- Fluor-546/594-coupled secondary antibodies (1:1000 dilution) (Molecular Probes) and images taken by confocal microscopy. Immunoprecipitation (IP) was performed on lysates from HeLa or MCF7-ErbB2 cells overexpressing ANXA2-RFP and S100A11-GFP (wild type or mutants) or only S100A11-turbo-GFP. Immuno complexes were captured with RFP-Trap agarose beads (Chromotek) or turbo-GFP antibody (OriGene) coupled to sepharose-G beads and washed 4 times before immunoblot analysis (Extended blots showing Co-IP are presented in Supplementary Figure 4).