Figure 4.

14‐3‐3ζ binds to phospho‐beclin 1^S295 in HCC‐LM3 and CSQT‐2 cells. Co‐IP was conducted in both (A) HCC‐LM3 and (B) CSQT‐2 cells. (C) Fragments (1371 bp) containing BECN 1 gene CDS were amplified from these cells and analysed with agar electrophoresis. The sequence information closely to RLPS²⁹⁵VP (R, arginine; L, leucine; P, proline; S, serine; V, valine) was shown in D,E. (F) Flag‐labelled beclin 1^S295 and beclin 1^S295A were transfected into CSQT‐2 cells, and co‐IP with anti‐flag antibody for IP and anti‐14‐3‐3ζ antibody for IB was performed. (G) HCC‐LM3 and (I) CSQT‐2 cells were infected with n.c. oe‐LV or 14‐3‐3ζ oe‐LV, and 72 h later, cells were treated with 20 mg/mL actinomycin D for additional 12 h. Protein levels of phospho‐beclin 1^S295 were assessed with Western blot. In addition, (H) HCC‐LM3 and (J) CSQT‐2 cells were infected with n.c. sh‐LV or 14‐3‐3ζ sh‐LV, and 72 h later, cells were treated with 20 mg/mL actinomycin D for additional 12 h under hypoxia. Protein levels of phospho‐beclin 1^S295 were also assessed with Western blot. β‐Actin was the reference. nc, negative control; LV, lentivirus; oe, overexpression; shRNA, short hairpin RNA