FIG 4.

Loss of Lats and YAP phosphorylation and increased TAZ levels with PCBP2 depletion. (A) Loss of Lats1 phosphorylation with PCBP2 depletion. PCBP2 was depleted in MCF10A cells, and the lysates were subjected to IP with anti-total Lats1 or normal rabbit IgG. Immune complexes and WCE were examined for phospho-Lats (pLats) and total Lats1 (tLats1) (left) and quantified (right). (B) Increased TAZ protein and decreased YAP phosphorylation with PCBP2 depletion. PCBP2 was depleted, and levels of phospho-YAP, total YAP, and TAZ were measured by Western blotting in duplicate (left) and quantified (right). (C and D) Lack of specific interaction between PCBP2 and Hippo-related transcripts. PCBP1 or PCBP2 was immunoprecipitated from MCF10A cells, and coprecipitating RNA was analyzed by RT-PCR. Rabbit IgG precipitations served as a negative control. The relative abundance of precipitated transcripts relative to IgG (C) or relative to total mRNA, normalized to actin (D), are shown. Normalization to GAPDH rather than actin yielded similar results. (E) Expression of TAZ and PCBP2 in breast cancer cell lines. MCF7 and MDA-MB-231 cells were grown under identical conditions, and the lysates were analyzed by Western blotting. (F). Increased TAZ protein with PCBP2 depletion in HEK293 cells. HEK293 cells were transfected with the indicated siRNAs and analyzed by Western blotting. (G) Increased TAZ in cells depleted of PCBP2 by alternative siRNA. MCF10A cells were transfected with control nontargeting siRNA or one of two siRNAs against PCBP2 (P2-1 or P2-2). TAZ or PCBP2 levels were detected by Western blotting. (H) TAZ mRNA levels with PCBP2 depletion. TAZ transcripts were measured by real-time PCR and are presented relative to the control. The experiments were replicated three times. The error bars indicate SEM. *, P < 0.0001.