Affinipure goat anti mouse fitc

Cells were fixed with 4% paraformaldehyde (Sigma Aldrich 158127, St. Louis, MI) in PBS for 15 minutes at room temperature and permeabilized with 0.1% Triton X‐100 (Sigma Aldrich X100, St. Louis, MI) in PBS for 5 minutes at room temperature. Samples were blocked for 30 minutes in PBS with 2% bovine serum albumin (BSA, Sigma Aldrich A2153, St. Louis, MI). Primary antibodies against β‐tubulin (GeneTex GTX11307, CA), HAS3 (Sigma Aldrich SAB2108148, St. Louis, MI), GFAP (GeneTex GTX108711, CA) and TUBB3 (GeneTex GTX631836, CA) were diluted 1:100 in PBS with 1% BSA and then incubated for 2 hours at room temperature. AffiniPure goat anti‐mouse‐FITC (Jackson ImmunoResearch 115‐095‐062, PA) and AffiniPure goat anti‐rabbit rhodamine (Jackson ImmunoResearch 111‐025‐003, PA) secondary antibodies were diluted 1:50 and incubated for 1 hour at room temperature. Coverslips were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories H‐1000, CA). FRET activity assay was measured by Leica FRET AB system and all the images were captured by confocal microscopy (Leica, Wetzlar, Germany).

Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Samples were blocked for 30 min in PBS with 2% bovine serum albumin (BSA). Primary antibodies against β-tubulin (GTX101279, GeneTex, Irvine, CA, USA), γ-tubulin (GTX113286, GeneTex, Irvine, CA, USA), acetyl-α-tubulin (GTX16292, GeneTex, Irvine, CA, USA), and HAS3 (SAB2101014, Sigma-Aldrich, St. Louis, MO, USA) were diluted 1:100 in PBS with 1% BSA, and then incubated with the cells for 2 h at room temperature. AffiniPure goat anti-mouse FITC (15-095-003, Jackson ImmunoResearch, West Grove, PA, USA) and AffiniPure goat anti-rabbit rhodamine (111-025-144, Jackson ImmunoResearch, West Grove, PA, USA) secondary antibodies were diluted 1:50. Slides were incubated with secondary antibodies for 1 h at room temperature. Samples were mounted with VECTASHIELD Antifade Mounting Medium (H-1000, Vector Laboratories, Burlingame, CA, USA) and imaged via confocal microscopy (DMI 6000B CS, Leica, Wetzlar, Germany).