Detecting PRDM9 in cell lines

Available cell lines were investigated for the presence of PRDM9 protein by gel electrophoresis followed by Western blotting with a commercially available PRDM9 antibody (Abcam, Cambridge, UK). To assess which cell model would be most appropriate; primary myoblast, primary fibroblasts and immortalised HEK293 cell lysates were run alongside a human testes (HuT) protein lysate sample (Figure 6.1). Testes tissue lysate was a kind gift from Professor David Elliot (Institute of Genetic Medicine, Newcastle University). After 60 minutes of exposure there was a faint signal detected in all sample lanes however there were three bands visible at ~90, ~65 and ~40 kDa. There was no protein detection in the human testes lysate using 4 µL of lysate of unknown concentration, however GAPDH signal was also low suggesting that there might not have been a high enough quantity of protein used to detect PRDM9.

Figure 6.1 Western blot detection of PRDM9 in different cell lines. Protein size marker was included (M), sizes are provided in kDa. 20 µg of myoblast, fibroblast and HEK293 cell lysate was loaded on a 4-20% bis-tris gel alongside 4 µL human testes lysate of unknown protein concentration (HuT). Membrane was incubated with anti-PRDM9 (1:500 dilution) or GAPDH (1:1000 dilution) overnight at 4°C.

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In order to determine whether the faint bands observed in Figure 6.1 were PRDM9, larger amounts of protein were required. HEK293 cells were chosen as the most appropriate model because immortalised cells typically divide at a much faster rate without replicative senescence and provide more cellular material than primary cell lines (Hayflick, 1965). Optimal protein concentration for detection was performed by running concentration gradients (Figure 6.2). Double the volume (8 µL) of human testis tissue lysate was used compared to the previous blot (Figure 6.2 A). There was a single band corresponding to a peptide slightly larger than the predicted molecular weight of 103 kDa in both the HEK293 and human testes sample lanes. There was also a band at ~60 kDa in the highest concentration HEK293 sample lanes. Detection of these bands was very weak and required long exposure times. Next, a higher protein concentration gradient of HEK293 cell lysate was tested (Figure 6.2 B). Bands were observed at ~100 and ~60 kDa but additional bands were also seen at ~120 and ~30 kDa. Thus, loading higher amounts of protein onto the gel gave more non-specific banding when using the anti-PRDM9 antibody to detect protein levels.

Figure 6.2 Western blot detection of PRDM9 in HEK293 cell and human testes lysate. Protein size marker was included on each blot (M), sizes are denoted in kDa. A) HEK293 cell lysate gradient from 40-5 µg protein was loaded on a 4-20% bis-tris gel alongside 8 µL human testes lysate of unknown protein concentration (HuT). B) HEK293 cell lysate gradient from 120-50 µg was loaded on a 4-20% bis-tris gel. Membrane was incubated with anti-PRDM9 (1:500 dilution) or GAPDH (1:1000 dilution) overnight at 4°C.

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Due to the embryonic nature of PRDM9 expression, the human embryonic stem cell line H9 (H9 hESC) was investigated for PRDM9 protein expression (Figure 6.3). The HEK293 cell lysate was immunoblotted alongside H9 as a comparison. As expected from previous results, HEK293 lysate gave bands at ~120 and ~60 kDa in size as well as faint bands at several other molecular weights. Bands were observed at ~60, ~50 and ~20 kDa in the H9 sample lanes. The band at ~120 kDa and a weaker band at ~80 kDa observed in the HEK293 samples were not present in the H9 samples. Overall, this suggests that the HEK293 and H9 cell lines have completely different protein expression profiles using the commercially available PRDM9 antibody in this study. Importantly, the band detectable at a molecular weight close to that of PRDM9 (103 kDa) was observed in the HEK293 sample lysate only.

Figure 6.3 Western blot detection of PRDM9 in HEK293 and H9_hESC cell lysates. Protein size marker was included (M), sizes are denoted in kDa. Protein concentration gradient for each sample lysate was loaded on a 4-20% bis-tris gel (100 – 25 µg). Membrane was incubated with anti-PRDM9 (1:500 dilution) overnight at 4°C.

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The well-established immortalised cell line Neuron-committed Teratocarcinoma (NT2) is derived from a human embryonic teratoma. As it is of embryonic origin, it was tested for PRDM9 protein expression by immunoblotting alongside three primary myoblast cell lines available in our laboratory (Figure 6.4). Detection of PRDM9 was unsuccessful in all cell lines tested, even after 1 hour of membrane exposure. Incubation of the samples with anti-βactin showed that protein was detectable in all sample wells further

confirming that PRDM9 was not present in these cell lines (Figure 6.4).

Figure 6.4 Western blot detection of PRDM9 in primary myoblast and NT2 cell lysates. Protein size marker was included (M), sizes are denoted in kDa. 75 µg sample lysate was loaded on a 4-20% bis-tris gel. Membrane was incubated with anti-PRDM9 (1:500 dilution) overnight at 4°C.

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To check antibody specificity, two antibodies against PRDM9 were kindly provided by the HPA (AlbaNova, Stockholm, Sweden). These antibodies were used for Western blot detection of PRDM9 in HEK293 cell lysate (Figure 6.5). Bands were detected at ~55, ~45 and ~25 kDa using the Abcam and HPA antibodies respectively (Figure 6.5Figure 6.5).

When compared to levels of β-actin detectable in the lysate (Figure 6.5) the bands detected using the Abcam and HPA059555 antibodies appeared to be very faint compared to HPA063372 (Figure 6.5). No band was observed at the predicted PRDM9 weight, 103 kDa, using any of the antibodies.

Figure 6.5 Western blot detection of PRDM9 in HEK293 cell lysate. 100 µg total protein was loaded in each sample well and electrophoresed on 4-20% bis-tris gels. Protein size marker was included on each blot (M), sizes are provided in kDa. A) PRDM9 protein was immunoblotted using either Abcam, HPA059555 or HPA063372 antibody against PRDM9. B) β-actin protein was immunoblotted for each sample lane as a loading control. Membrane was incubated with anti-PRDM9 (1:500 dilution) or anti-βactin (1:1000) overnight at 4°C.

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The peptide sequence used to generate the antibody against PRDM9 was commercially available (Abcam, Cambridge, UK). This peptide comprises amino acids 432-481 of the human PRDM9 protein sequence and could therefore be used for antigen binding assays to elucidate which of the protein bands detected by blotting could potentially be PRDM9. Briefly, HEK293 cell lysate was incubated with purified PRDM9 antigen peptide,

Western blotted and compared to untreated HEK293 total lysate. The strongest band at ~55 kDa in the total cell lysate sample lane was blocked in the sample incubated with the antigen (Figure 6.6, red arrow).

Figure 6.6 Western blot detection of PRDM9 in HEK293 cell lysate blocked with 1 µg/mL antigen peptide. Protein size marker was included on each blot (M), sizes are provided in kDa. Total lysate: 100 µg total HEK293 cell lysate was loaded and electrophoresed on a 4-20% bis-tris gel (left). Blocked lysate: after incubation with PRDM9 peptide antigen, 100 µg HEK293 cell lysate was loaded and electrophoresed on a 4-20% bis-tris gel (right).

Together, these data suggest that the protein observed at ~55 kDa is the specific target of this antibody. Although this does not confirm that full length human PRDM9 is being

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detected, the protein target at ~55 kDa is detectable in HEK293 cell lysate using this antibody.