Anti prdm1

Cells were lysed with the lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 20 μM MG132, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 μg/ml leupeptin, and 2 μg/ml pepstatin). A total of 500 μg of lysate protein was incubated with the primary antibodies at 4°C overnight to immunoprecipitate the protein complexes. Immune complexes were then collected by direct binding to protein A-Sepharose. The immunoprecipitates were then blotted with the corresponding antibody against the protein as indicated. Anti-Prdm1 (Cell Signaling), anti-Baf170 (Cell Signaling), anti-pStat1 (Cell Signaling), anti-Stat1 (Cell Signaling), anti-Snf5 (Santa Cruz), anti-Pias1 (Santa Cruz), anti-p50 (Cell Signaling), anti-IgG (Santa Cruz), and anti-Elf3 (Santa Cruz) were used for IP/co-IP analysis.

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before [36 (link)–39 (link)]. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-LXRα (Abcam, ab41902), anti-ELL2 (Proteintech, 12727-1), anti-MSC (Santa Cruz, sc293482), anti-KDM2A (Abcam, ab191387), anti-PRDM1 (Cell Signaling Tech, 9115), anti-Nrf2 (Cell Signaling Tech, 12721), or pre-immune IgG.

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).