Figure S5.
MST and CAP Experimental Controls
a, Typical MST traces. The relative fluorescence is plotted against the experiment time. Each trace corresponds to a sample with a different concentration of AQP4 whereas calmodulin (CaM) concentration remained constant, except for the trifluoperazine (TFP) titration experiment (bottom right) in which TFP was varied and both AQP4 and CaM were kept constant. The difference in relative fluorescence before (blue column) and after (red column) heating is used to calculate ΔFnorm. The position of the red column was determined by the M.O. Affinity Analysis software (Nanotemper) as the time interval that gave the best signal to noise ratio; b, Recombinant AQP4 is a functional water channel. Fluorescence traces from a proteoliposome shrinking assay showing that liposomes containing purified AQP4 (blue) have significantly higher water permeability than empty liposomes (gray). The increase in fluorescence corresponds to the fluorophore ((5)6-carboxyfluorescein) present on the inside of the liposomes becoming more fluorescent as the liposomes shrinks when mixed with hyperosmotic solution. The data were fitted to a double exponential function (solid blue and black lines for AQP4-containing and empty liposomes, respectively) and the rate constant (k) was used to calculate the osmotic water permeability (Pf). For AQP4-containing liposomes Pf = 5.9 ± 0.5 × 10−2 cm/s and for control liposomes Pf = 1.2 ± 0.2 × 10−2 cm/s, corresponding to an AQP4 single channel permeability of 1.1 ± 0.1 × 10−13 cm3/s. Analysis by t test showed a statistically-significant difference between AQP4-containing liposomes and empty liposomes (p = 0.0015; Table S2). Related to Figure 3; c, Representative Spike 2 software processed CAP traces. A normal short-latency negative-positive CAP trace was observed in sham animals which was ablated in DC+vehicle- and DC+PKCi-treated rats, but partially restored in DC + CaMi and DC + PKAi-treated rats. Following a dorsal hemi-section in the same animals at the end of the experiment, CAP traces were ablated in all animals, indicating DC axon regeneration. Related to Figure 4.