Issues with RNF43 antibodies to reliably detect intracellular location

Abstract

RNF43 is an important negative regulator of β-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of β-catenin. RNF43 has also been suggested to regulate β-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/β-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers.

Fig 1. Epitope location and generation of DLD-1 RNF43 ΔEX8-9 clone.

A. Schematic representation of RNF43 mRNA and epitope location of RNF43 antibodies. The ab217787 antibody was raised against a N-terminal epitope encoded by exons 2 and 3, while the other three antibodies were raised against epitopes encoded by exons 8 and 9. A DLD-1 cell clone was generated that entirely misses these exons leading to a p.(Glu284_Pro769delext*56) deletion on protein level. B. Confirmation of correct deletion of exons 8 and 9 on DNA level. Left panel shows PCR with primers flanking the deletion. The expected approximate 900bp fragment is observed in the ΔEX8-9 clone, while the original 4kb fragment is too big to be amplified. Middle and right panels show, respectively, PCRs for exons 8 and 9, leading to the expected 283 and 914bp fragments in the wild-type cell line, whereas only non-specific bands are observed in the ΔEX8-9 clone. DNA marker used is the 1kb DNA ladder from Promega (#G5711) C. Confirmation of correct deletion of exons 8 and 9 on mRNA level. Primers flanking exons 8 and 9 reveal the expected 1904 and 445bp fragments, respectively, for the wild-type cells and ΔEX8-9 clone. D. A quantitative RT-PCR analysis of RNF43 exons 8–9 shows undetectable levels in the ΔEX8-9 clone. Interestingly, as shown by a qRT-PCR for exons 6–7, total RNF43 levels are decreased about 200-fold in this clone. In conclusion, we have successfully generated a DLD-1 clone that shows strongly reduced levels of RNF43 mRNA entirely lacking exons 8 and 9.

Fig 2. Immunoblot analysis of RNF43 antibodies.

The HPA008079, ab84125, 8D6 and ab217787 antibodies cannot specifically recognize endogenous RNF43 by immunoblotting. No signal is expected in the HCT116, DLD-1 ΔEX8-9, and KM12 RNF43 KO lanes for the exon 8–9 located antibodies, while ab217787 may detected 17 and 37 kDa truncated bands in, respectively, HCT116 and DLD-1 ΔEX8-9. All antibodies may detect a specific truncated band in the DLD-1 and KM12 WT lanes. However, only non-specific bands are observed. The 8D6 and HPA008079 antibodies are able to detect overexpressed RNF43 (lanes 1). The dashed lines demarcate a non-essential protein marker and/or sample lane that were removed from the image. The table at the bottom shows the expected protein bands that can be detected for each sample. Protein sizes are based on transcript ID ENST00000407977.7 and protein ID CCDS11607.1. Overexpressed RNF43 is marked with a red asterisk. Original images can be found in S1 Fig.

Fig 3. Immunofluorescence analysis of RNF43 antibodies.

DLD-1, DLD-1 ΔEX8-9, and HCT116 cells were cultured on glass slides and stained with HPA008079, ab84125, ab217787and 8D6 antibodies. DLD-1 ΔEX8-9 and HCT116 cells are not expected to reveal any staining, but in all cases signals are observed comparable with the wild-type control DLD1. DAPI was used to stain the nuclei. For the 8D6 antibody the DAPI-staining is coincidentally stronger in the DLD-1 ΔEX8-9 cells, giving the potential false impression that the green 8D6 signal is weaker in these cells compared with their wild-type controls. However, evaluation of multiple independent images shows that non-specific signals are of comparable intensity. Larger images, a higher intensity image for ab84125, and negative control test are shown in S2 Fig. Scale bar, 25um.

Fig 4. RNF43 antibodies can detect overexpressed RNF43 using immunofluorescence.

HCT116 cells were transiently transfected with HA-tagged RNF43 and simultaneously stained with each RNF43 antibody and a HA-tag antibody. DAPI was used to stain the nuclei. Overexpressed RNF43 was present in the cytoplasm and detectable with all antibodies to some extent, except for the ab217787 antibody. Scale bar, 25um.

Fig 5. Immunohistochemical analysis of RNF43 antibodies.

The indicated cell lines were formalin-fixed and embedded in paraffin. HCT116 cells transiently overexpressing RNF43 were also included. All antibodies give the same non-specific staining pattern as observed in the IF-experiments, showing that they all strongly recognize a non-specific protein not being RNF43. The 8D6 and HPA008079 antibodies can detect overexpressed RNF43, indicated by arrows, while this is not the case for ab84125 and ab217787. Scale bar, 50um.

Fig 6. Immunoblot analysis and IF of 3xFLAG RNF43 knockin clones.

A. Immunoblot analysis of Caco-2 and OE19 clones with a 3xFLAG knockin at the C-terminus of RNF43. A sensitive ECL Ultra Western HRP Substrate system was required to reveal the bands. A 30x diluted sample of RNF43-FLAG transfected HEK293T cells is added as control. B. Using a Tyramide SuperBoost system a small number of 3xFLAG positive OE19 cells could be identified. No positive cells were observed in the parental OE19 cell line. The staining pattern resembles that of overexpressed RNF43 showing a cytoplasmic and perinuclear pattern, the latter reminiscent of an ER location. However, given the low number of positive cells in only one cell line, we cannot draw a reliable generalized conclusion about RNF43’s intracellular location. Scale bar, 25um.