Ab103021

IFN-γ (315-05), IL-4 (214-14), M-CSF (315-02) and GM-CSF (315-03) were purchased from Peprotech (Rocky Hill, NJ). LPS (from Escherichia coli, serotype 055:B5, L4005) was purchased from Sigma (St. Louis, MO). The primary antibodies and dilutions used were: Flag (Sigma, F3165, 1:2000), α-tubulin (Sigma, T6199, 1:2000) and β-actin (Sigma, A2228, 1:5000), β-TrCP(Cell Signaling Technology, 4394, 1:1000), Nesprin1 (Santa Cruz, sc-99065, 1:500), Nesprin2 (Santa Cruz, sc-365097, 1:500), LaminA/C (Santa Cruz, sc-7292, 1:1000), ubiquitin (Santa Cruz, sc-8017, 1:1000), CD11b (eBioscience, 17-0112, 1:100), CD86 (eBioscience, 11-0862, 1:100), CD45 (eBioscience, 48-0451, 1:100), F4/80 (eBioscience, 25-4801, 1:100), CD206 (R&D systems, FAB2535P, 1:100) and Arg1 (R&D systems, IC5868P, 1:100) were obtained from. human SUN1 (Abcam, ab103021, 1:1000), mouse SUN1 (Abcam, ab124770, 1:1000) and mouse SUN2 (Abcam, ab124916, 1:1000), and mouse SUN2 (Abcam, ab198981, 1:100, FCS). An antibody specific for human SUN2 (1:1000) was produced by Shanghai Immune Biotech (Shanghai, China).

The spermatocyte spreads and seminiferous tubules squashes were blocked for 30 min with 1% BSA and 0.05% Tween in PBS (PBS-T) and then were incubated overnight at 4 °C with the following primary antibodies: Sycp3 (Abcam, ab15093 and ab97672, 1:800; and Santa Cruz, sc-20845, 1:20); Sycp1 (Abcam, ab15087, 1:800) Dmc1 (Santa Cruz, sc-22768, 1:50); Trf1 (Alpha Diagnostics, TRF12-S, 1:50); Sun1 (Abcam, ab103021, 1:50); H1t (gift from Dr Mary Ann Handel, The Jackson Laboratory, Bar Harbor, Maine, USA, 1:200) H3K9-3me (Milipore, 05-1250, 1:2000); γH2AX (Milipore, 05-636, 1:300); Mlh1 (Becton Dickinson, 551091, 1:100); Rad51 (Santa Cruz, sc-8349, 1:100) Terb-1 (affinity-purified rabbit antiserum, 1:100)34 (link) and Cdk2 (Abcam, ab7954, 1:50). RingoA was detected using the R55A mouse monoclonal antibody12 (link), which recognizes the sequence NDHQSNK corresponding to amino acids 283-289 (hybridoma supernatant, 1:1). The slides were then washed in blocking buffer and incubated for 1 h at 37 °C with appropriate Alexa Fluor-labelled secondary antibodies (Invitrogen, A21442, A31571, A21441, A21468, A21203, A11017, 1:300). Finally, the slides were washed and mounted in ProLong Gold antifade reagent (Molecular Probes).
For telomere FISH staining, Telomere PNA FISH kit/cy3 (Dako, K5326) was used on 16 dpp testis spreads following the protocol described by the manufacturer.

Rabbit polyclonal anti-SUN1 antibodies (ab103021) were from Abcam, rabbit polyclonal Phospho-(Ser) PKC Substrate Antibody (#2261) was from Cell signaling. GFP was detected by mAb K3-184-2^{21 (link)}. For Nesprin-2, mAb K20-478 and pAbK1 were used^{18 (link), 19 (link)}. Mouse monoclonal anti-NXF1, mouse monoclonal anti-Nup153 and rabbit anti-LaminB1 polyclonal antibodies were from Abcam, mouse monoclonal anti-GAPDH-POD used in western blots was from Sigma. mAb K3-184-2 was used to detect GFP in western blots^{24 (link)}. Membranes were stripped of antibodies by incubation with 0.1 M NaOH for 5 min for two times and reused for immunolabelling. The antibodies were used at concentrations recommended by the supplier.