PUF60 (HMV4728)

PUF60 is a member of the Pumilio family of RNA-binding proteins which is coded by the PUF60 gene on chromosome 8q24.3. PUF60 is involved in various cellular processes, including mRNA metabolism, splicing regulation, and assembly of RNA-protein complexes. It plays a critical role in the regulation of gene expression through its ability to bind to RNA, thus influencing the stability, translation, and localization of target mRNAs. PUF60 is especially important for neural development and organogenesis. PUF60 Knock-Out Mice display embryonic lethality. PUF60 germ line mutations have been linked to specific syndromes, including neurodevelopmental disorders that affect cognitive functions and behavior. Evidence is accumulating that PUF60 also plays an important role in cancer biology, for example through its role as a c-myc transcriptional repressor. Aberrant PUF60 expression has been linked to increased tumor aggressiveness and poor prognosis in several malignancies. Its role in cellular transformation and tumor progression it not fully elucidated yet.

In normal tissues, PUF60 is ubiquitously expressed. Tissues with a rather low PUF60 expression include hepatocytes, syncytiotrophoblast cells of the placenta, as well as superficial epithelial cell layers of the intestine and squamous epithelium. Spermatids in the testis are PUF60 negative.

Images describing the PUF60 staining pattern in normal tissues obtained by the antibody HMV4728 are shown in our “Normal Tissue Gallery”.

These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression PUF60) where a rather ubiquitous expression is described (endothelial cells occur in all tissues).

Positive control = Testis: All cell types (except spermatids) should show strong nuclear PUF60 staining.

Negative control = Testis: Spermatids should be PUF60 negative.

Normal tissue gallery

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV4728 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• How does PUF60 depletion affect cellular differentiation and function in normal and diseased tissues?
• What role does PUF60 play in the molecular pathways involved in cancer?
• How does PUF60 expression correlate with tumor phenotype in specific cancer types?

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody HMV4728 specificity is in line PUF60 RNA expression data in normal tissues which were collected in three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, and which are summarized in the Human Protein Atlas (Tissue expression PUF60). In agreement with HMV4728 immunostaining data, PUF60 RNA expression was found in all tissues at comparable levels. lymphoid tissues. However, orthogonal validation is not optimal for assessing ubiquitously expressed proteins.

Comparison of antibodies: True expression of PUF60 in all cell types with nuclear PUF60 positivity by HMV4728 is corroborated by an identical nuclear staining obtained by a commercially available independent second antibody (termed “validation antibody”). Most of all, both antibodies showed several characteristic variabilities in their nuclear staining patterns which included a negativity of spermatids in the testis and a low staining intensity in hepatocytes, syncytiotrophoblast cells of the placenta, as well as in superficial cell layers of the intestinal epithelium and of squamous epithelium.

Normal tissue gallery