Figure 7. Hypoxia recruits one SUMO1 monomer to each cell surface NaV1.2 channel.
Single CFP-NaV1.2 or CFP-NaV1.2-Lys38Gln (K38Q, blue) channels and SUMO1 tagged with mCherry (m-SUMO1, red) were studied in CHO cells by TIRFM as described in the Materials and methods. Data represent 5–8 cells in each case and biophysical parameters and single particles statistical analyses are summarized in Supplementary file 1a and Table 2, respectively. (a) Left, single co-localized particles with both mCherry and CFP fluorescence were observed at the surface of cells expressing NaV1.2 and SUMO1. Simultaneous continuous photobleaching time courses revealed complexes to have one subunit of each type. (b) Histogram of photobleaching steps showing that hypoxia increased single mCherry-SUMO1 (red) subunits at the cell surface co-localized with NaV1.2 channels (blue), without a change in subunit stoichiometry (Table 2). (c) Histogram of photobleaching steps showing that SENP suppresses hypoxia-induced increase in single mCherry-SUMO1 (red) subunits at the cell surface co-localized with NaV1.2 (blue). (d) Histogram of photobleaching steps showing that hypoxia does not increase in single mCherry-SUMO1 (red) subunits at the cell surface co-localized with NaV1.2-Lys38Gln channels (blue).