Anti p50

All the proteins were extracted using RIPA buffer containing PMSF (Beyotime, China). The protein samples were loaded onto the SDS-PAGE gels and electrotransferred to PVDF membranes (Milipore, America). The membranes were blocked with 5% nonfat milk for 2 hours, and incubated overnight at 4°C with specific primary antibodies, i.e., anti-hTERT (1:1000, Abcam, USA), anti-KLF4 (1:1000, Abcam, USA), anti-p65 (1:1000, Abcam, USA), anti-p50 (1:1000, Abcam, USA), anti- CDX2 (1:1000, Abcam, USA), anti-MUC2 (1:1000, Abcam, USA) and anti-GAPDH (1:500, Boaoshen, China) antibodies. The membranes were subsequently incubated with a secondary antibody. The bands were quantified using Quantity One software (Bio-Rad, USA), and the gray value was analyzed using GraphPad Prism software (GraphPad, USA).

Lysates, cytosolic and nuclear extracts were electrophoresed on a 4-12% polyacrylamide gel (Invitrogen) and proteins were transferred to an Immobilon membrane (Millipore). Membranes were blotted with anti-IκBα (Cell Signaling, #4814), anti-IκBβ (R&D systems, AF5225), anti-p50 (Abcam, ab7971), anti-p65 (Cell Signaling, #8242), anti-IL-1α (R&D systems, AF-400-NA), anti-HDAC (for nuclear extracts, Cell Signaling, #5356), anti-Calnexin (for lysate, Enzo Life Sciences ADI-SPA-860), and anti-GAPDH (for cytosolic extracts, Cell Signaling, #5174). Densitometric analysis was performed using Image Studio (LiCor).

Nuclear extracts were isolated using the standard approach (73 (link), 79 (link), 80 (link)). Briefly, cells were grown to 80% confluence and then exposed to C. parvum for various times. Cells were treated with EDTA (Sigma) and washed with PBS, and the cell pellet was resuspended in 1 ml of cold buffer A (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, and 1 mM dithiothreitol [DTT]). Nuclear pellets were isolated from the whole-cell protein by centrifugation at 14,000 rpm for 1 min at 4°C and resuspended in cold buffer B (20 mM HEPES, 1.5 mM MgCl₂, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, and 1 mM DTT) with vigorous agitation in the cold room for 30 min. The supernatant containing nuclear proteins was collected after centrifugation at 14,000 rpm for 5 min at 4°C. Protein concentration of each nuclear extract or whole-cell lysate was determined and subsequently analyzed by Western blotting. The following antibodies were used for blotting (details in Table S1): anti-Brg1 (Santa Cruz), anti-H3K36me3 (Abcam), anti-H3K4me3 (Abcam), anti-Stat1 (Cell Signaling), anti-pStat1 (Cell Signaling), anti-Pias1 (Santa Cruz), anti-Snf5 (Santa Cruz), anti-Baf170 (Cell Signaling), anti-p50 (Abcam), anti-Elf3 (Santa Cruz), anti-Cdc42 (Fisher Scientific), anti-Actin (Sigma), and anti-Prdm1 (Cell Signaling).

Chromatin immunoprecipitation analysis was accomplished using the Magna ChIP™ A kit (Millipore). In brief, QKI5-silenced RAW 264.7 cells and control cells were incubated with medium or LPS (1 µg/ml) for 4 h, crosslinked in 1% formaldehyde for 10 min at room temperature and quenched with unreacted formaldehyde by the addition of 0.125 M glycine. Cells were washed twice with PBS and suspended in cell lysis buffer containing protease inhibitors, incubated on ice for 15 min, and centrifuged at 800 × g for 5 min. The pellet was homogenized in nuclei lysis buffer containing protease inhibitors and subjected to sonication on ice, followed by incubation overnight at 4°C with anti-Ahr (Thermo Fisher, clone RPT9), anti-p50 (Abcam), anti-p65 (Merck), anti-STAT1 (CST), normal mouse IgG, or normal rabbit IgG in the presence of protein A magnetic beads. The reactions were incubated overnight at 65°C to reverse the cross-links. DNA was purified by spin column and analyzed by PCR with the following primers: 5′-CGATGCTAAACGACGTCACATTGTGCA-3′ and 5′-CTCCAGAGCAGAATGAGCTACAGACAT-3′, specific for the κB site in the IL-6 promoter.