2Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland
The centrosome is a highly organized cellular organelle that functions as the main microtubule-organizing center (MTOC) of animal cells and is crucial for fundamental cellular processes (1). Proteomic analyses have revealed a network of interactions among centrosomal proteins (2, 3). We report here that two widely used commercial mouse monoclonal antibodies against the master cell cycle regulator Cdk1 (sc-54, Santa Cruz Biotechnology, Dallas, TX; ab18, Abcam, Cambridge, United Kingdom) cross-react with the centrosomal protein Cep152. Our results provide an explanation for previous conflicting reports on the localization of Cdk1 and also question the reported centrosomal recruitment of Cdk1 by Cep63.
Recently, we reported the existence of a Cep57-Cep63-Cep152 centrosomal complex, in which Cep63 and Cep152 exhibit a strong mutual dependency for their localization at centrosomes (4). Given that Cep63 has been reported to be required for the presence of the master cell cycle regulator Cdk1 at centrosomes (5, 6), we expected that centrosomal Cdk1 localization should also depend on Cep152 function. To test this hypothesis, we carried out pull-down experiments, performing Western blot analysis of the fusion proteins purified from HEK293 cel l s expres sing SNAP-Cep152, SNAP-Cep63, SNAP-Cep57, or Cdk1-SNAP. SNAP-tag is a 20 kD protein tag that can be covalently labeled (e.g. with fluorescent dyes), thus permitting detection of the fused proteins in vitro and in vivo (7-9). We probed the membrane with the mouse monoclonal anti-Cdk1 antibody sc-54 that was utilized in the study reporting the dependency of centrosomal Cdk1 on Cep63 function (5). This antibody has also been used in over 200 published studies (www.scbt.com/datasheet-54-cdc2-p34-17-antibody.html).
Surprisingly, we found that sc-54 recognized not only Cdk1, but also Cep152 (Figure 1). To clarify this issue, we purchased four other commercial anti-Cdk1 antibodies that were raised against other Cdk1 immunogens and repeated the Western blot analysis (Table 1, Figure 1, Supplementary Figure S3B). We found that the mouse monoclonal anti-Cdk1 antibody ab18 similarly cross-reacted with Cep152, whereas the rabbit polyclonal HPA003387 antibody or mouse monoclonal antibodies ab8040 and 610038 did not (Figure 1).
The observed cross-reactivity of some anti-Cdk1 antibodies does not pose a significant problem in simple Western blotting experiments because of the size difference between Cdk1 and Cep152, but it could potentially cause misleading results in immunofluorescence and pull-down experiments. We therefore set out to perform immunofluorescence with all five antibodies to determine whether the cross-reactivity observed by Western blot also applies to this experimental approach, which was previously used to report the reliance of centrosomal Cdk1 on Cep63 function (5). We fixed control cells and cells depleted of Cdk1 or Cep152, using two distinct siRNAs/siLNAs corresponding to each protein. The resulting samples were then stained with anti-Cdk1 antibodies (one antibody per sample), along with anti-γ- tubulin antibodies to mark centrosomes. In each case, we quantified the resulting centrosomal signal intensity in cells in late G2/early mitosis relative to the γ-tubulin signal; the outcome of these experiments is summarized in Table 1, and more detailed results are listed in Supplementary Table S1. First, we found that both sc-54 and ab18 antibodies labeled centrosomes in control conditions throughout the cell cycle, as reported previously for sc-54 (10). These results contrast with earlier studies using PSTAIR and CTR453 antibodies, which led to the conclusion that Cdk1 localizes to centrosomes solely in late G2/early mitosis (11). In accordance with the latter result, we found that in control conditions the HPA003387, ab8040, and 610038 antibodies indeed significantly labeled centrosomes solely during late G2/early mitosis (Supplementary Figure S1A). Second, we found that the centrosomal signal of the sc-54 and ab18 antibodies decreased substantially upon depletion of Cep152 but not upon depletion of Cdk1 (Table 1, Supplementary Table S1, Supplementary Figure S1B). Conversely, we found little or no change in the centrosomal signals for the HPA003387, ab8040, or 610038 antibodies upon depletion of Cep152, but a substantial decrease was observed upon depletion of Cdk1 (Table 1, Supplementary Table S1). Analogous results were obtained when Cep63 was depleted by RNA interference (Supplementary Figure S1B), as expected given that Cep63 and Cep152 are mutually dependent for their centrosomal localization (4).
Next, we conducted confocal imaging of U2OS cells to precisely localize the centrosomal signal of anti-Cdk1 antibodies relative to C-Nap1, which marks the proximal end of the centriole, or Cep152, which is present in a ring just above the proximal end (6, 12-15). The confocal microscopy experiments established that both sc-54 and 610038 antibodies labeled the proximal end of the centriole (Supplementary Figure S2A and B). Interestingly, however, we found that the distribution of the sc-54 signal was different from that of the 610038 signal, with the former matching the localization of the Cep152 ring perfectly (Supplementary Figure S2C and D). To confirm this, we generated a U2OS cell line expressing a Cdk1-SNAP fusion protein. Immunofluorescence experiments showed that the Cdk1-SNAP signal resembled the staining obtained with the 610038 antibody and not the sc-54 antibody (Supplementary Figure S2E and F). To investigate whether the distribution of Cep152 and Cdk1 at centrosomes may differ, we performed STED (stimulated emission depletion) microscopy. In accordance with previous observations (4), super-resolution microscopy showed that Cep152 was present at the proximal end of the parental centriole in a ring-like structure 367 nm in diameter on average (Supplementary Figure S3A). By contrast, we found that the 610038 antibody signal was positioned in a much more diffuse manner, most likely in the pericentriolar material (PCM), and could not be resolved as a ring by STED microscopy (Supplementary Figure S3A).
Finally, we found that there may be a simple explanation for the observed cross-reactivity between Cdk1 and Cep152 using the sc-54 and the ab18 antibodies. Indeed, these antibodies were raised against partially overlapping immunogens of Cdk1, which exhibit some degree of similarity with the amino acid sequence of Cep152 (Supplementary Figure S3B and C).
In conclusion, our observations indicate that both sc-54 and ab18 antibodies recognize not only Cdk1 but also Cep152 in Western blot and immunofluorescence assays. By contrast, HPA003387, ab8040, and 610038 appear to recognize Cdk1 specifically, and demonstrate that Cdk1 localizes most likely in the PCM of centrosomes in a cell-cycle dependent manner. Our work also serves as a reminder that numerous independent control experiments are needed to verify antibody specificity in immunofluorescence and other antibody-based experiments (16).
The authors thank Fernando Romero Balestra, Meritxell Orpinell and Rūta Gerasimaitė for their useful comments on the manuscript.
The authors declare no competing interests.
Address correspondence to Kai Johnsson, Ecole Polytechnique Fédérale de Lausanne, Institut des sciences et ingénierie chimiques Lausanne, Switzerland, E-mail: [email protected]; or to Pierre Gönczy, Ecole Polytechnique Fédérale de Lausanne, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland, E-mail: [email protected]
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