Ab198981

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 total proteins were extracted from cells using radioimmunoprecipitation (RIPA) cell lysis buffer (BB-3209, BestBio Co., Ltd., Shanghai, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrically transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with the sealing solution for 1 h, followed by incubation at 4 °C overnight with the addition of following primary antibodies: rabbit polyclonal antibody to CD133 (1: 1000, ab198981), CD13 (1: 500, ab154116), Nanog (1: 500, ab80892), SOX2 (1: 1000, ab97959), OCT-4 (1: 1000, ab19857), rabbit monoclonal antibodies to STAT3 (1: 1000, ab68153) and p-STAT3 (1: 2000, ab76315) (Abcam Inc., Cambridge, MA, USA), and rabbit polyclonal antibody to CADM1 (1: 1000, A1892, ABclonal Biotech Co., Ltd., Cambridge, MA, USA). On the following day, the membrane was incubated with HRP conjugated goat anti-rabbit immunoglobulin G (IgG) (1: 5000, A21020, Abbkine, USA) at 37 °C for 1 h and developed with ECL reagent. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference, the relative protein levels of target proteins were expressed as the ratio of gray value of target band to that of internal reference band. Each experiment was conducted 3 times.