Amotl1 hpa001196

The primary antibodies of TEAD1 (sc-376113), TEAD4 (sc-134071), and CTGF (L-20) (sc-14939) were from Santa Cruz (Dallas, TX, USA). YAP1 (ab52771) antibody and HA tag (ab18181) were achieved from Abcam (Cambridge, MA, USA). AMOTL1 (HPA001196) and Flag-tag (F3165) antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other primary antibodies were from Cell Signaling (Danvers, MA, USA), including p21 (#2946), p27 (#2552), pRb (Ser807/811) (#9308), Erk (#9102), pErk (#9101), Myc (#2278), cyclin D1 (#2978), c-Myc (#9402), and GAPDH (#2118). Anti-Mouse IgG-HRP (Dako, Glostrup, Denmark, 00049039, 1:30,000) and anti-Rabbit IgG-HRP (Dako, 00028856, 1:10,000) were used for secondary antibodies. The related protocol was suggested before [13 (link)].

Cells were lysed in non-denaturing ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM EGTA, 1% (v/v) Triton X-100, pH 7.6) supplemented with cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ phosphatase inhibitors (Roche Applied Science). Lysates were cleared for 10 min at 14,000×g at 4 °C. Subsequently, 500 μg protein was incubated with 1 μg primary antibody. The following antibodies were used: β-Catenin (8480, Cell Signaling), VE-cadherin (2500, Cell Signaling), AMOTL1 (HPA001196, Sigma-Aldrich), AMOTL2 (23351-1-AP, Proteintech), YAP1 (PA1-46189, Thermo Fischer Scientific), and TAZ (560235, BD Biosciences) overnight at 4 °C with gentle rocking. Subsequently, protein A/G magnetic beads (88803, Thermo Fisher) were added for an additional 2 h. Beads were collected using magnetism and extensively washed with lysis buffer. Beads were then heated for 5 min at 95 °C in denaturing sodium dodecyl sulfate buffer before western blot analysis.