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. 2018 Jan 3;293(7):2395–2407. doi: 10.1074/jbc.RA117.000861

Figure 3.

Figure 3.

SUN5 directly interacts with DNAJB13. A, GST pulldown of SUN5-interacting proteins in testes. The SUN domain of mouse SUN5 was purified and immobilized on GST–beads and then incubated with testis lysate. The eluates were resolved by SDS-PAGE and stained using a Colloidal Blue staining kit. The asterisks indicate the bands that were specifically pulled down by GST–SUN5 beads and retrieved for analysis by mass spectrometry. GST protein was used as a negative control. B, SUN5 interacted with DNAJB13 in the yeast two-hybrid assay. SUN5C (coiled-coil domain and the SUN domain of SUN5) and the SUN domain, instead of full-length SUN5, interacted with DNAJB13 in the yeast two-hybrid assay. SUN1C and KASH5 LR were used as a positive control. Empty vector was used as a negative control. C, GST–SUN5 pulldown results were confirmed with immunoblotting of endogenous DNAJB13. GST pulldown was repeated as in A, but the eluates were analyzed with immunoblotting against DNAJB13 following SDS-PAGE. The analysis showed that DNAJB13 could be detected in the GST–SUN5 pulldown lane but not in the GST alone lane. D, mouse polyclonal DNAJB13 antibody detected overexpressed MYC–DNAJB13 and co-localized with MYC signals in HeLa cells. MYC–DNAJB13 was overexpressed in HeLa cells, and immunofluorescence staining was performed using rabbit anti-MYC antibody (green), mouse anti-DNAJB13 antibody (red), or a combination of the two antibodies, DNA was stained with DAPI (blue). The DNAJB13 signal was detected only in the overexpressed cells (middle panel) and overlapped with the MYC signal, indicating the specificity of murine polyclonal DNAJB13 antibody. The boxed areas have been enlarged in the lower row. E, murine polyclonal DNAJB13 antibody detected purified His—FLAG–DNAJB13 and endogenous DNAJB13 in the testis. 500, 100, and 10 ng of purified His—FLAG–DNAJB13 and 20 μg of adult testis and ovary extracts were loaded and detected by murine DNAJB13 antibody and rabbit FLAG antibody. Ovary extract was used as a negative control. FLAG antibody was used as a positive control for immunoblotting. F, SUN5 bound DNAJB13 in the testis. Endogenous co-immunoprecipitation of SUN5 and DNAJB13 from testis lysate was performed with murine DNAJB13 antibody, and SUN5 was detected with rabbit anti-SUN5 antibody.