With highly specific ABfinity recombinant monoclonal antibodies

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The Hippo signaling pathway is an evolutionarily conserved pathway that has been shown to play a critical role in controlling organ size through the regulation of both cell proliferation and apoptosis. Dysregulation of the Hippo pathway results in aberrant cell growth and neoplasia. Given its involvement in these vital cell processes, it is not surprising that mutations in key Hippo pathway proteins are linked to a variety of cancers [1,2].

At the cellular level, the Hippo pathway integrates signals through several mechanisms, including G protein–coupled receptor (GPCR) signaling and the apicobasal polarity fundamental to epithelial cell function [3]. Kinase cascade and nuclear transcription modules form the backbone of the Hippo pathway. The kinase cascade includes serine/threonine kinases such as mammalian STE20–like protein kinases (MST1/2) and large tumor suppressors (LATS1/2), along with adaptor proteins including Salvador homolog 1 (SAV1), MOB kinase activator 1A (MOB1A), and MOB kinase activator 1B (MOB1B). The kinase cascade functions to restrict the activity of two transcriptional coactivators—Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)—which are further modified by several Hippo regulators.

Antibodies are essential to the study of pathway proteins. However, for many components of the Hippo pathway, specific antibodies are either unavailable or poorly characterized. To address this need, Thermo Fisher Scientific has made a concerted effort to develop highly specific, application-tested antibodies directed against key proteins in this pathway.

Antibodies for kinase cascade proteins

In the kinase arm of the Hippo pathway, MST1/2-mediated phosphorylation of SAV1 and MOB1A/B leads to the recruitment, phosphorylation, and subsequent activation of LATS1/2. In conjunction with MOB1, the activated LATS1/2 kinases phosphorylate YAP/TAZ. These phosphorylation events result in the cytoplasmic sequestration and degradation of YAP/TAZ, mediated by 14-3-3 proteins. Figures 1A and 1B demonstrate the specificity of Invitrogen anti-MST1/2 and anti-SAV1 antibodies in western blot and immunofluorescence/immunocytochemistry (IF/ICC) applications using siRNA-mediated knockdown. In response to nocodazole treatment, LATS2 translocates from the cytoplasm into the nucleus, then binds to and activates p53, inducing LATS2 expression [4]. Figure 1C shows the specificity of the Invitrogen anti- LATS2 antibody in IF/ICC using this nocodazole-induced upregulation of LATS2 expression.

Figure 1. Antibodies directed against proteins in the Hippo pathway kinase cascade. (A) The specificity of Invitrogen MST2 Antibody (clone 19H19L39), ABfinity Rabbit Monoclonal was determined by western blot analysis using siRNA-mediated MST2 knockdown in A549 cells. (B) The specificity of Invitrogen SAV1 Antibody (clone 6H5L16), ABfinity Rabbit Monoclonal was demonstrated by immunofluorescence/immunocytochemistry (IF/ICC) using siRNA-mediated SAV1 knockdown in HCT116 cells. (C) The specificity of Invitrogen LATS2 Antibody (clone 17H14L2), ABfinity Rabbit Monoclonal was demonstrated by IF/ICC; increased LATS2 expression was observed in U2OS cells treated with nocodazole. For IF/ICC, primary antibodies were detected with Invitrogen Goat Anti–Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 (green), nuclei were stained using Invitrogen ProLong Diamond Antifade Mountant with DAPI (blue), and cytoskeletal F-actin was labeled with Invitrogen Rhodamine Phalloidin. Chemiluminescence detection was performed using Invitrogen Goat Anti–Rabbit IgG (H+L) Secondary Antibody, HRP (0.25 μg/mL, 1:4,000 dilution) and Invitrogen Novex ECL Chemiluminescent Substrate Reagent Kit on the Invitrogen iBright FL1000 Imaging System.

Antibodies for transcription factors and Hippo regulators

In the nuclear transcription module of the Hippo pathway, several DNA-binding proteins regulate the transcription of genes encoding Hippo pathway proteins and also undergo modification themselves. When not phosphorylated, YAP and TAZ translocate to the nucleus and serve as transcriptional coactivators for Hippo pathway genes. Their most significant interacting partners are the TEA-domain family member transcription factors (TEAD1–4) [5]. Figure 2A shows the specificity of the Invitrogen anti-TEAD4 antibody in western blot analysis using siRNA-mediated knockdown. Another transcription regulator, VGLL4, selectively binds to TEAD and interferes with the YAP–TEAD interaction. The specificity of the Invitrogen anti-VGLL4 antibody has been demonstrated with IF/ICC using siRNA-mediated knockdown (Figure 2B).

Angiomotin proteins (AMOT), which play a central role in tight junction maintenance, are also important negative regulators of YAP/TAZ, either by binding YAP/TAZ or by promoting their inhibitory phosphorylation. Phosphorylated AMOT (ser176) is downregulated during serum starvation, a characteristic that was used to show the specificity of the Invitrogen anti-AMOTpS176 antibody in IF/ICC (Figure 2C). In contrast to AMOT, 53BP2 (ASPP2) is a positive regulator of YAP, facilitating the dephosphorylation of YAP/TAZ. Specificity of the Invitrogen anti-53BP2 antibody has been demonstrated in IF/ICC using siRNA-mediated knockdown (Figure 2B).

WWC1 (KIBRA), a mechanical regulator that associates with tight junctions and other cell polarity complexes, can induce the phosphorylation of LATS1/2 [6]. The specificity of the Invitrogen anti-WWC1 antibody is shown in IF/ICC using siRNA-mediated knockdown (Figure 2B). The Hippo kinase cascade can also be initiated by TAO kinases, which phosphorylate MST1/2 [7]. TAO kinases, including TAO3 and JIK, are differentially expressed in androgen-dependent and -independent cell lines, a feature that was used to show the specificity of the Invitrogen anti-JIK antibody (Figure 2D).

Figure 2. Antibodies that recognize Hippo pathway regulators. (A) The specificity of Invitrogen TEAD4 Polyclonal Antibody was determined by western blot analysis using siRNA-mediated TEAD4 knockdown in HeLa cells. (B) The specificity of Invitrogen 53BP2 Antibody (clone 8H3L19), ABfinity Rabbit Monoclonal and Invitrogen WWC1 Antibody (clone 1H4L22), ABfinity Rabbit Monoclonal was demonstrated by immunofluorescence/immunocytochemistry (IF/ICC) using siRNA-mediated knockdown in Caco-2 cells. The specificity of Invitrogen VGLL4 Antibody (clone 16H12L24), ABfinity Rabbit Monoclonal was demonstrated by IF/ICC using siRNA-mediated VGLL4 knockdown in HCT116 cells. (C) The specificity of Invitrogen Phospho-AMOT (Ser176) Antibody (clone 18H4L17), ABfinity Rabbit Monoclonal was demonstrated by IF/ICC using untreated and serum-starved HEK293 cells, which exhibit reduced AMOTpS176 expression. (D) The specificity of Invitrogen JIK Antibody (clone 1HCLC), ABfinity Rabbit Oligoclonal was demonstrated by IF/ICC to observe the differential basal expression of JIK in androgen-dependent (LNCaP) and -independent (DU145) cells. Fluorescent and chemiluminescent labeling reagents are detailed in Figure 1.

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