*4.7. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)*

In order to quantify the expression levels of ELAV2, ELAV3, and ELAV4 in the DRG neurons from STZ-sensitive, STZ-resistant and control CD-1 mice subgroups, the total RNA was extracted from dissociated ganglia using the GenElute Mammalian Total RNA MiniPrep Kit (#RTN70, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. RNA concentrations were determined by spectrophotometric measurements at 260 and 280 nm (Beckman Coulter DU 730). In agreement with the manufacturer guidelines (Sigma-Aldrich, St. Louis, MO, USA) for the GenElute™ Mammalian Total RNA Miniprep Kit, in our experiments the A260:A280 ratio was 2.038 ± 0.07. Reverse transcription was performed using the High-Capacity cDNA Archieve Kit (Applied Biosystems, Foster City, California, USA). The relative abundance of ELAV transcripts was assessed by qRT-PCR using TaqMan methodology and the ABI Prism 7300 Sequence Detection System (Applied Biosystems). Reactions were carried out for 35 cycles in triplicate. *Elavl2* (#Mm00516015\_m1), *Elavl3* (#Mm01151962\_m1), *Elavl4* (#Mm01263580\_mH) and the mouse control assay for glyceraldehydes-3-dehydrogenase (*Gapdh*, #Mm999999\_g1) were obtained from Life Technologies (Carlsbad, CA, USA), and used in accordance with manufacturer's guidelines. From each animal group, 3 animals were sacrificed for the qRT-pCR analysis, and each gene was analyzed in triplicate. Quantitative RT-PCR data for each *Elavl* were normalized with *Gapdh* mRNA levels and relative amounts of mRNA were determined using the comparative cycle thresholds [57].

#### *4.8. Immunofluorescence*

DRG neurons in primary culture seeded on 13-mm coverslips were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and immunostained. Non-specific binding was blocked with donkey serum (#017-000-001, Jackson ImmunoResearch Laboratories, UK). DRG neurons were incubated with the primary antibodies overnight at 4 ◦C. We have used the following primary antibodies: rabbit polyclonal IgG anti-ELAVL2 antibody (1:100; #ab96471, Abcam, Cambridge, UK), rabbit polyclonal IgG anti-ELAVL3 antibody (1:100; #ab78467, Abcam), and rabbit polyclonal IgG anti-ELAVL4 antibody (1:100; #ab96474, Abcam), considering the target

specificity of these antibodies described by previous studies [20,25,58,59]. Then, DRG neurons were incubated with the secondary antibody donkey polyclonal anti-rabbit IgG (H+L) conjugated with Rhodamine Red X (1:100; #111-295-003, Jackson ImmunoResearch Laboratories, UK) for 1h at room temperature. The primary antibody was omitted in negative-control samples. Images were captured using a confocal fluorescence microscope (LSM 710, Carl Zeiss, Oberkochen, Germany) equipped with a 63× oil objective. The following acquisition parameters were used: pinhole corresponding to 1 Airy Unit, 62 μm for the 488 nm laser, digital gain of 1.00, and 5% intensity of the laser, as we previously employed [60]. The acquisition parameter settings were kept fixed across all the image acquisition sessions. Image acquisition was carried out using Zeiss LSM Image Browser software (Carl Zeiss, Germany).
