NOVA1 protein, human

Dcc genomic DNA that spans exons 16 and 17 (5.6 kb total) was PCR amplified from mouse spinal cords and cloned into the pDEST26 gateway vector containing a CMV promoter (Thermo Fisher). Dcc minigene was transfected into HEK293T cells together with the splicing factors or an empty vector at a 1:1 ratio. Cells were cultured for 48 hr and the total RNA was collected using Trizol (Thermo Fisher). Reverse transcription was carried out from a T7 promoter (present in pDEST26) using SMARTScribe reverse transcriptase (Clontech, Mountain View, CA), and semi-quantitative PCR was performed to amplify multiple isoforms. Point mutations were introduced by PCR reactions using Pfu polymerase (Agilent, Santa Clara, CA), and were confirmed by DNA sequencing. A V5 tag at the C-terminus of NOVA1, NOVA2, and PTBP2 was used to confirm protein expression using western blotting.

All
electrochemical experiments were performed on an Autolab PGSTAT302N
potentiostat from Metrohm equipped with the FRA32 module and operated
with Nova 1.11.2 software. Each measurement was conducted on a freshly
cleaved HOPG surface. Before the experiment, the CE was flame-cleaned
with a blue butane flame and the RE was thoroughly washed with DI
water. To avoid the contamination of the HOPG by the adsorption of
air-bound hydrocarbons, a phenomenon well established in the literature
for the basal plane of graphite,^{27 (link)} the
solution was deposited on the WE within 1 min of cleaving the surface.
Unless specified otherwise, the applied potential, E, throughout the main text is referred vs Ag/AgCl_(sat. KCl) (see Section S1.2 in the SI). The experimental
protocol used for the static measurements was composed of consecutive
potential pulses from 0 to −2 V vs Ag/AgCl wire with a step
of 50 mV. For the positive side, the same approach was followed in
the potential range between 0 and +1.5 V vs Ag/AgCl wire. The duration
of the pulses was adjusted accordingly (varying from 5 to 15 s) for
each electrolyte concentration in the range of 0.1–16 m for
KF and 0.1–20 m for CsF based on preliminary dynamic experiments
for the determination of the time required to attain equilibrium;
the latter was indicated by the plateau in the calculated CA values.
A similar strategy was adopted for the dynamic measurements, in which
the potential was directly stepped from 0 to either −2 or +1.5
V vs Ag/AgCl wire three consecutive times. Each repetition represents
one cycle. Once again, the duration of the potential pulse was chosen
based on the required time needed (0.5 s) to ensure a steady state
response for the electrolyte concentration used. For the investigation
of the surface processes occurring during cathodic and anodic polarization
in different electrolyte concentrations, cyclic voltammetry (CV) experiments
were carried out over a potential range from 0 to −2 and 0
to +1.5 V vs Ag/AgCl_(sat. KCl), respectively, at a
scan rate of 1 V s^–1. Electrochemical impedance
spectroscopy (EIS) measurements were performed in the frequency range
between 20 kHz and 10 Hz, using an imposed AC rms amplitude of 7 mV
peak-to-peak. The EIS experimental data was evaluated for its compliance
with Kramers–Kronig (KK) criteria by fitting the AC response
of the system to the admittance representation of a theoretical circuit
containing a ladder of n RC elements in series, with
an additional capacitance and/or inductance in parallel to the ladder
structure, using the software developed by Boukamp.^{28 (link)} The choice of the aforementioned equivalent circuit relies
on the blocking nature of the electrodes under study (the impedance
increases to infinity as frequency approaches zero), which renders
the Voigt-type approximation inappropriate.^{28 (link)} The compliance with KK criteria was assured for all data by the
values of the relative residuals, calculated to be less than 0.5%
for both the real and imaginary parts of the impedance and the chi-square
parameter which was found to be on the order of 10^–7 for the complete data series. All of the experiments were conducted
inside a faraday cage.

Voltammetry experiments were performed with an Autolab
PGSTAT204
potentiostat running on Nova 2.1 (Metrohm Nordic AB, Sweden). We used
the following electrodes purchased from Metrohm Nordic AB (Sweden):
screen-printed carbon electrode (DRP-150), glassy carbon working electrode
(GCE; diameter of 5 mm, RDE.GC50.S), Pt electrode (6.0331.010), and
a Ag/AgCl single-junction reference electrode (EQCM. refEL.S). A Mo
plate was also used as an electrode (Sigma-Aldrich, 357200-25.6G).
Electrochemical measurements in “beaker configuration”
were performed using a three-electrode system with the GCE as the
working electrode (WE), the Ag/AgCl single-junction reference electrode
(RE), and the Pt rod as the counter electrode (CE). For the experiments
conducted in the microfluidic cell (Figure 2a), three types of electrodes were prepared
and/or utilized. Electrode 1 was the DRP-150 with a carbon-based WE
modified with a film of electropolymerized PANI (150 CV scans in 0.1
M aniline/0.5 M H₂SO₄). Electrode 2 was the
Mo plate modified with a custom-made screen-printed carbon electrode
(SPCE). Electrode 3 was an unmodified DRP-150. More details about
the preparation of the electrodes are presented in the Supporting
Information. Figure S1 presents a real
picture of the device.
The fluidic cell was designed in AutoCAD
2020 and printed with
an Ultimaker 3 3D printer (Ultimaker B.V., the Netherlands) using
PLA filament (Ultimaker, the Netherlands). The rubber spacers (thicknesses
of 0.5 and 1 mm) were purchased from (Ecoflex, USA) and cut to an
appropriate size to create the microfluids for the cell. The cell
is composed of two internal compartments: the first compartment is
situated between Electrode 1 and Electrode 2, while the second is
situated between Electrode 2 and Electrode 3. The WEs of actuator
1 and actuator 2, the sensor, and (a common) RE are placed in the
first compartment, while the common CE (the carbon part of the DRP-150;
Electrode 3) is placed in the second compartment. The Ag element in
the DRP-150 in Electrode 1 acts as the common RE. The PANI-C element
of Electrode 1 acts as the WE of actuator 1 and acidifies the sample
upon the application of 0.4 V for 300 s. The Mo element in Electrode
2 acts as the WE of actuator 2 and delivers MoO₄^2– and H⁺ by
applying a current of 0.15 mA for 300 s. Finally, the C path in Electrode
2 acts as the WE of a DIP sensor that uses either CV or SWV. The potential
was switched in the cathodic direction to ensure the partial reduction
of Mo(VI) centers in the phosphomolybdate complex to Mo(V).