Anti sumo 1

Fourty adult animals were transferred to an Eppendorf tube containing 20 μl of water. 20 μl of 2xSDS buffer were added, and the sample was incubated for 5 min at 95°C. Genomic DNA was digested by adding 1 μl of DNase (Qiagen) and incubating for 5 min at room temperature, followed by a 5 min inactivation at 95°C. Proteins were separated by SDS PAGE on 4–12% acrylamide gradient gels and blotted onto PVDF membranes. After blocking non-specific binding sites with 5% milk or bovine serum albumin in TBST (20 mM Tris, 150mM NaCl, 0.1% Tween 20), the membranes were incubated with the primary antibody diluted in TBST containing 5% milk overnight at 4°C. After incubation with HRP-conjugated secondary antibodies, the protein bands were viualized by chemiluminescence using the SuperSignal West Pico or Dura Chemiluminescent Substrate (Thermo Scintific). Quantification was performed by measuring the band intensities using Fiji’s measurement tools [56 (link)]. Band intensities were first normalized to the alpha-tubulin loading controls and then to the maximum value in each experiment. The following antibodies were used: anti-SUMO-1 1:500 (S5446 Sigma), anti-Flag 1:3000 (Sigma F3165-1MG), anti-Tubulin 1:10 000 (Abcam ab18251), HRPGoat anti-Rabbit 1:2000 (Jackson ImmunoReserach 111-035-144) and HRP Goat anti-Mouse 1:2000 (Jackson ImmunoReserach 115-035-146).

About 1.42 × 10¹⁰ vgs of AAV vectors were loaded onto a denaturing SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gel. Resolved proteins were further transferred into polyvinylidene fluoride (PVDF) membrane (Pall Corporation, Port Washington, NY). Subsequently, the membrane was blocked with 5% bovine serum albumin (BSA) for 1 h. Membranes were then probed with anti-AAV (B1) (1:500; Fitzgerald, North Acton, MA) or anti-SUMO-1 (1:1,000; Sigma–Aldrich) primary antibodies and detected with an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,500; Abcam, Milton, Cambridge, United Kingdom). The signals were developed by chemiluminescent substrate (SuperSignal™ West Pico PLUS; Thermo Scientific). Densitometric quantification was performed by using ImageJ^{35 (link)} in three different blots with two measurements at least for each blot developed.