Sab2101014

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).

For protein extraction, cells were washed twice with ice-cold PBS and lysed on ice in Golden lysis buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 5.95 mM EDTA, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, and 10% glycerol) supplemented with protease inhibitors (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Specific antibodies against HAS3 (SAB2101014, Sigma-Aldrich, St. Louis, MO, USA), p21 (GT8611, GeneTex, Irvine, CA, USA), α-tubulin (GT114, GeneTex, Irvine, CA, USA), ATG5 (GTX113309, GeneTex, City, CA, USA), LC3 (GTX127375, GeneTex, Irvine, CA, USA), and GAPDH (sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA) were diluted 1:2000 in Tris-buffered saline/Tween 20, and the membranes were incubated for 2 h at room temperature. Horseradish peroxidase-conjugated anti-mouse IgG (sc-2354, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) secondary antibodies were diluted 1:4000 and incubated with the membranes for 1 h at room temperature.