Rabbit anti β tubulin antibody

To determine tubulin distribution, FaDu cells were seeded on glass cover slips for 24 h. Cells were treated with WMJ-J-09, paclitaxel or colchicine for another 24 h. Cells were then washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After permeabilization for 30 min in 0.1% Triton X-100 in PBS, FaDu cells were washed twice and incubated with 1% BSA in PBS for another 1 h. To observe tubulin distribution, cells were reacted with rabbit anti-β-tubulin antibody (Cell Signaling Technology, Danvers, MA, United States) (1:100 dilution in PBS) for 16 h at 4°C. Slides were washed twice and incubated with FITC-conjugated goat anti-rabbit IgG for another 1 h. Slides were mounted with DAPI containing mounting solution (SlowFad Gold, Thermo Fisher Scientific, Waltham, MA, United States) and then observed under a confocal microscope (Zeiss, LSM 410). Green fluorescence indicated β-tubulin, and blue fluorescence (derived from DAPI) represented nuclei.

Xiaoyao Pills (XYW) (TaiJi, China), fluoxetine hydrochloride (FLX) (Patheon, France) and Amitriptyline hydrochloride (Sigma, USA) were dissolved in 0.9% saline to prepare solutions. Paeoniflorin (C₂₃H₂₈O₁₁), Liquiritin (C₂₁H₂₂O₉), Glycyrrhizic acid (C₄₂H₆₂O₁₆), Ligustilide (C₁₂H₁₄O₂) (Cheng Du Ai Fa, China). LPS (Escherichia coli 055:B5) was provided by Sigma (St. Louis, MO). Rabbit anti-TrkB antibody (1:1000; Cell Signaling Technology; Cat. No. #4603), rabbit anti-cAMP response element-binding protein (CREB) antibody (1:1000; Cell Signaling Technology; Cat. No. #9197S), rabbit anti-p-CREB antibody (1:1000; Cell Signaling Technology; Cat. No. #9198S), rabbit anti-β-Tubulin antibody (1:1000; Cell Signaling Technology; Cat. No. #2128). Rabbit anti-brain-derived neurotrophic factor (BDNF) antibody (1:1000; Abcam; Cat. No. #ab108319). Rabbit anti-DLG4, PSD95-specific, polyclonal antibody (1:1000; Proteintech; Cat. No. #20665-1-AP) and rabbit anti-synaptophysin polyclonal antibody (1:1000; Proteintech; Cat. No. #17785-1-AP). Rabbit anti-GAPDH antibody (1:1000; Servicebio; Cat. No. #GB11002). Rabbit anti-IL-6 polyclonal antibody (1:300; Servicebio; Cat. No. #GB11117) and Alexa Fluor 488 goat anti-rabbit IgG (1:400; Servicebio; Cat. No. #GB25303).

Due to the lower capability for dendritic outgrowth, compared with axons, the central region of 2 mm × 20-mm silicone barrier area normally contained axons only. Microtubule-associated protein 2 (MAP2, a specific marker for dendrites and neuronal somata) and microtubule-associated protein Tau (a specific marker for axons and neuronal somata) were used to identify neurites in the axon-only area. Cells were labeled with either monoclonal mouse anti-MAP2 antibody (1:2,000; clone HM-2, Sigma-Aldrich, St. Louis, MO, United States) or Tau (1:1,000; clone Tau46, Sigma-Aldrich, St. Louis, MO, United States), followed by Alexa Fluor 594 goat anti-mouse IgG1 (1:100 Molecular Probes, Thermo Fisher Scientific, Rochester, NY, United States). Detailed methods are described below in the section relating to immunocytochemistry. To test the purity of our neuronal cell cultures, cultured cells were double labeled with polyclonal rabbit anti-β-tubulin antibody (1:500, Cell Signaling, Danvers, MA, United States) and a neuronal cell maker (either MAP2 or Tau). Alexa Fluor 594 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG were used as corresponding secondary antibodies. A high ratio of MAP2 to Tau-positive cells compared to β-tubulin III-positive cells demonstrated the purity of the cultured neurons.

Rabbit polyclonal anti-MuLV p30 antiserum was described previously [13 (link)]. Mouse monoclonal anti-HIV-1 p24^CA antibody (YDHIVgp24) was purchased from MyBioSource (San Diego, CA, USA). β-Tubulin was used for the loading control in western blots and was detected by rabbit anti-β-Tubulin antibody (Cell Signaling Technology, Danvers, MA, USA). For western blots, we used an anti-mouse IgG antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA) and an anti-rabbit IgG antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA). The antibodies for LC3 (Cell Signaling Technology) and p62/SQSTM1 (Proteintech Group, Inc., Rosemont, IL, USA) were used to check autophagic flux in the cells expressing Arf6Q67L. To examine the role of Arf6 in MuLV release, the following chemical inhibitors were used: UNC3230 (MedChemExpress, Monmouth Junction, NJ, USA), ML299 (Aobious Inc., Gloucester, MA, USA), FIPI (Tocris, Minneapolis, MN, USA), LY294002, wortmannin, rapamycin, and bafilomycin A1 (Cayman Chemical, Ann Arbor, MI, USA). MG132 and lactacystin were purchased from AdipoGen Life Sciences (San Diego, CA, USA).

INS‐1 cells were harvested with ice‐cold lysis buffer supplemented with complete proteinase inhibitor mixture (Roche). Other specific information of Western blotting assay was performed as previously described previously.¹⁹ The antibodies used are as follows: rabbit anti‐CASK antibody (1:1000; Cell Signaling), rabbit anti‐DNMT1 (1:1000; Cell Signaling), mouse anti‐DNMT3a (1:1000; Santa Cruz Biotechnology), rabbit anti‐DNMT3b (1:1000; Abcam), rabbit anti‐Akt(pan) antibody (1:1000; Cell Signaling), rabbit anti‐p‐Akt Ser473 antibody (1:5000; Abcam), rabbit anti‐iNOS Antibody (1:1000; Cell Signaling) or rabbit anti‐β‐tubulin antibody (1:4000; Cell Signaling).