Journal: Cells

Article Title: Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

doi: 10.3390/cells10030567

Figure Lengend Snippet: Evidence for Ca 2+ -dependent interaction between annexin A2 (ANXA2) and S100A13 under serum-free conditions. ( A ) Pull-down assay using Strep -tagII-S100A13. Strep -tagII-S100A13 and C6 glioma cell lysates were incubated in the absence (left lane) and presence (right lane) of 100 μM Ca 2+ . Results represent the immunoblot using anti-ANXA2 IgG (upper lanes) and anti-β-actin antibody (lower lanes). The pull-down assay in A showed that ANXA2 dimer interacts with Strep -tagII-S100A13 in C6 glioma cell lysates in a Ca 2+ -enhanced manner. In the previously published study , we have shown that p40 Syt-1 also interacts with Strep -tagII-S100A13 in C6 glioma cell lysates in a Ca 2+ -enhanced manner. As both pull-down assays using Strep -tagII-S100A13 have been done with the same C6 glioma cell lysates, it appears that S100A13 forms the same protein complex with both experiments p40 Syt-1 and ANXA2. The experiment was done using same C6 glioma cell lysates as previously reported to show the interaction between Strep- tagII-S100A13 and p40 Syt-1 . ( B ) Serum-free-induced release of S100A13, but not ANXA2 from C6 glioma cells. Results represent the time course of protein levels of S100A13, ANXA2 and β-actin in cells (upper panels) and conditioned medium (CM) (lower panel) by immunoblot analysis. Extracellular S100A13 was recovered from CM using immunoprecipitation. ( C ) Schematic model of interaction between His 6 -ANXA2 and Strep -tagII-S100A13 bound to Streptavidin plate in an ELISA-based protein binding assay. Inset: enhancement of the interaction in the presence of His 6 -p40 synaptotagmin-1 (Syt-1). ( D , E ) No effect of amlexanox (Amx) on the His 6 -ANXA2 binding to Strep -tagII-S100A13 in terms of analyses of His 6 -ANXA2 concentration-dependency ( D ) and its reciprocal plot ( E ). ( F ) His 6 -p40 Syt-1 concentration-dependent enhancement of His 6 -ANXA2 binding to Strep -tagII-S100A13 in the presence of Ca 2+ . Data are presented as the mean ± standard error of the mean (S.E.M.) from a Tukey–Kramer multiple comparison test. * p < 0.05 and ** p < 0.01. n = 4 experiments per group.

Article Snippet: For immunoblotting, immunoprecipitation, immunocytochemistry or intracellular antibody delivery experiments, the following antibodies were used: mouse anti-prothymosin alpha (ProT α ) monoclonal antibodies (Clones: 2F11 and 4F4, Alexis Biochemicals, Lausen, Switzerland), rat anti-ProT α monoclonal antibody (Clone: 1 – 21) (developed in our laboratory, Nagasaki University, Nagasaki, Japan) [ ], N-terminus recognition goat anti-annexin A2 (ANXA2) polyclonal antibody and horseradish peroxidase (HRP)-conjugated mouse anti-β-actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-S100A13 antibody (kindly provided by Dr. T. Maciag, Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, USA), mouse anti-synaptotagmin-1 (Syt-1) monoclonal antibody (Wako, Osaka, Japan), mouse monoclonal anti-Stx-1 antibody (Wako, Osaka, Japan), rabbit anti-ATP8A2 polyclonal antibody (Abnova, Taipei, Taiwan), and normal mouse or goat IgG (ICN/Cappel Inc., Durham, NC, USA).

Techniques: Pull Down Assay, Incubation, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Protein Binding, Binding Assay, Concentration Assay