Gt114

For Western blot analysis, cells were lysed in 1 × RIPA buffer (Sigma). Western blot analysis were performed from three independent transfections using FLAG-tagged APOBECs and HA-tagged A1CF. α-Tubulin: internal loading control. The lysates were then subjected to Western blot with anti-FLAG M2 mAb (F3165, Sigma, 1:3000), anti-HA mAb (HA.C5, Abcam, 1:3000), and anti-α-tubulin mAb from mouse (GT114, GeneTex, 1:5000) as primary antibodies. Cy3-labelled goat-anti-mouse mAb (PA43009, GE Healthcare, 1:3000) was subsequently used as a secondary antibody. Cy3 signals were detected and visualized using Typhoon RGB Biomolecular Imager (GE Healthcare).

In situ hybridization was performed at a hybridization temperature of 62 and 65 °C for SSC-washes with riboprobes against xirp2a^{20 (link)} and her7^{10 (link)}. Whole-mount immunofluorescence was used to visualize F-actin with fluorescently tagged phalloidin (Thermo Fisher Scientific, dilution 1:2000) and antibodies against acetylated tubulin (T6793, Sigma Aldrich) and alpha tubulin (GT114, Genetex), each at a dilution of 1:1000.

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.