Anti green fluorescent protein

For immunostaining, the hind limb muscles were excised, formalin-fixed, paraffin embedded, and sectioned (5 μm thick). Blood vessels were stained with anti-CD31 (BD), anti-α-smooth muscle actin (SMA), or anti-green fluorescent protein (GFP) (Abcam Plc., Cambridge, MA, USA) antibodies, and then incubated with goat anti-rat Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 568 secondary antibodies (Life Technologies, Carlsbad, CA, USA). The sections mounted with VECTASHIELD medium containing DAPI and were observed under a laser scanning confocal microscope (Olympus FluoView FV1000; Olympus, Tokyo, Japan). Capillary and blood vessel densities were counted by identifying CD31- or α-SMA-positive vascular structures. Four randomly selected microscopic fields from 3 serial sections in each tissue block were examined per limb by 2 independent observers who were blinded to the experimental conditions.

Radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland) was used to lyse spinal cord or brain samples for western blot assays, while a non-denaturing lysis buffer [1% NP40, 5 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl (pH 8), 150 mM NaCl] containing the same inhibitors was used to lyse samples for co-immunoprecipitation. Samples were then separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–12% gels (Cat# V900859; Sigma-Aldrich, St. Louis, MO, USA). Protein bands were detected using enhanced chemiluminescence-Prime (Amersham, Buckinghamshire, UK) reagent. Densitometric analyses were conducted with Image Lab software (Bio-Rad, Hercules, CA, USA). Antibodies used for these analyses included anti-glyceraldehyde-3-phosphate dehydrogenase (mouse; 1:5000; Cat# GB12002; Servicebio), anti-CRMP3 (Cat# bs-11821R; 1:200; Bioss), anti-spastin (Cat# ab77144; 1:1000; Abcam), anti-horseradish peroxidase-conjugated goat anti-rabbit (1:5000; Cat# AS014; Abclonal Technology, Woburn, MA, USA), anti-horseradish peroxidase-conjugated goat anti-mouse (1:5000; Cat# AS003; Abclonal Technology), and anti-green fluorescent protein (GFP; 1:2000; Cat# ab290; Abcam). The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated at room temperature for one hour.

Chromatin immunoprecipitation (ChIP) was described elsewhere¹⁷ (link) with the following modifications. Briefly, HepG2 cells were transfected using X-tremeGENE transfection reagent (Roche Applied Science, Milan, Italy) with plasmid encoding Flag-tagged CREB3L3-N. Forty-eight hours after transfection, cells were treated with 1 mmol/L 8Br cAMP for 8 hours and fixed for formaldehyde cross-linking and ChIP. Protein–DNA complexes were immunoprecipitated overnight using the following antibodies: anti-Flag (Sigma-Aldrich), anti-PPARGC1A (anti-PGC1A; Santa Cruz Biotechnology, Dallas, TX), or anti-green fluorescent protein (GFP) (Abcam, Cambridge, UK) as negative control.