Sc 23950

Proteins in the adipocytes were extracted using Pro-Prep (Intron Biotechnology, Seongnam, Korea), and 20 μg of each protein was separated by SDS-polyacrylamide gel (12%) electrophoresis. Separated proteins were transferred to polyvinylidene difluoride (PVDF, Millipore, Burlington, MA, USA) membranes via a semi-dry transfer (Bio-Rad, Hercules, CA, USA). The membranes were blocked with non-fat dry milk for 1 h and incubated with the indicated primary antibodies (dilution 1:2000) overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h (Abcam, Cambridge, UK). HRP signals reacting with chemiluminescence reagents (Abclon, Guro, Korea) were detected on AGFA x-ray film CP-Bu NEW and quantified using ImageJ. Anti-acetylated α tubulin (Santa Cruz Biotechnology, SC-23950, Dallas, TX, USA) and anti-tubulin (Santa Cruz Biotechnology, SC-32293, Dallas, TX, USA) were used for the immunoblotting assay.

Antibodies against alpha-tubulin (ab15246) and KLF2 (ab139699) were obtained from Abcam (Cambridge, MA). Antibodies against acetylated-alpha-tubulin (sc23950) were obtained from Santa Cruz (Dallas, TX). Actin (A2066) antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Procedures were performed as previously described^{16 (link)}.

For immunoblotting and immunofluorescence, antibodies against Hsp90α (ab74248, Abcam), Hsp90β (ab2927, Abcam), GFP (sc-9996, Santa Cruz Biotechnology), acetylated α-tubulin (sc-23950, Santa Cruz Biotechnology), α-tubulin (T5168, Sigma or MAB1864, Merck), GM130 (610822, BD Pharmingen), Rab8 (610844, BD Pharmingen), MAP4 (11229-1-AP, Proteintech), MAP1B (HPA022275, Sigma), Flag (F1804, Sigma), HA (H6908, Sigma), and GAPDH (G8795, Sigma) were used. Horseradish peroxidase-conjugated secondary antibodies, DAPI solution, and nocodazole were from Sigma. Alexa-Fluor 488-, 546-, or 647-conjugated secondary antibodies, anti-fade mounting medium, and Alexa Fluor 647 Phalloidin were from Molecular Probes/Invitrogen. TSA (S1045) was obtained from Selleck. Protease, phosphatase inhibitors, and Anti-Flag or HA affinity gel were from Biomake. Anti-GFP-magnetic agarose (D153-10) was from MBL.

Sections of DRG or sciatic nerve from WT or Nogo-A KO rats, with or without CFA or SNI model, were used in this experiment. Sections of embedded tissue were collected using a cryostat, dried, and hydrated with PBS for 20–30 min at room temperature. 0.3% Triton x-100 in PBS was used for perforation at room temperature for 20 min. Sections were washed 3 times with PBS and incubated in the primary antibody overnight at 4 °C. The slides were washed with PBS and incubated with the fluorescent secondary antibody or dye at room temperature for 1 h or stored at 4 °C overnight. The primary antibody used: ace-α-tubulin (1:10,000, sc23950; Santa Cruz Biotechnology), pCRMP2 T514 polyclonal antibody (1:1000, ab62478; Abcam), guinea pig anti-TRPV-1 polyclonal antibody (AB5566; MilliporeSigma, Burlington, MA, USA), and TPPP3 (1:1000, bs-17172R, Bioss, Massachusetts, USA). The corresponding secondary antibodies for ace-α-tubulin, TRPV1, TPPP3, and pCRMP2 were FITC-conjugated goat anti-mouse IgG (1:200; Jackson ImmunoResearch, West Grove, PA, USA), TRITC-conjugated goat anti-guinea pig IgG (1:200, Thermo Fisher Scientific, MA, USA), TRITC-conjugated goat anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA), TRITC-conjugated donkey anti-rabbit IgG (1:200; Thermo Fisher Scientific, MA, USA), respectively.

All rats were deeply anesthetized with 10% chloral hydrate (0.3 g/kg, i.p.). The nitrocellulose membranes were incubated overnight at 4 °C with the primary antibody. The following commercially available antibodies were used as primary antibodies: ace-α-tubulin (1:10,000, sc23950, Santa Cruz Biotechnology, Dallas, Texas, USA), pCRMP2 T514 polyclonal antibody (1:1000, ab62478; Abcam, Waltham, MA, USA) guinea pig anti-TRPV-1 polyclonal antibody (1:1000, AB5566; MilliporeSigma, Burlington, MA, USA), GADPH (1:2000, TA-08, Zhongshan Golden Bridge Biotechnology, Guangdong, China), and β-actin (1:2000, TA-09, Zhongshan Golden Bridge Biotechnology, Guangdong, China). The blots were washed 3 times in Tris-buffered saline-Tween. The membranes were incubated with horseradish–conjugated secondary antibody (1:2000, goat anti-rabbit, rabbit anti-goat, rabbit anti-guinea pig, or goat anti-mouse; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature, and developed with a chemiluminescence kit (sc-2048, Santa Cruz Biotechnology).