Gtx100675

The ChIP assay was performed after cross-linking cells using formaldehyde. DNA was sonicated using an Ultrasonic Processor (Bioruptor UCD-200) at 12 cycles of 30 s with 30 s interval between cycles. Sonicated DNA was then centrifuged at 20,000 × g at 4 °C. Supernatant was used for ChIP assay using MAGnify ChIP system (Invitrogen). Two micrograms of mouse IgG, HDAC1 (Abcam-ab15050), H3ac (active motif 39139), C/EBPβ (Genetex GTX100675) was used per ChIP. Immunoprecipitated DNA was analyzed by qPCR, and Ct values were used to calculate the percentage of input enrichment.

Marc-145 cells or pig tissues were lysed in RIPA buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Jiangsu, China). The antibodies and their dilutions were shown as follows, anti-CEBPB (GTX100675, GeneTex, Alton Pkwy Irvine, CA, USA, 1:1000), anti-PRRS virus Nucleocapsid (GTX129270, GeneTex, Alton Pkwy Irvine, CA, USA, 1:1000), anti-β-tubulin (GB11017B, Servicebio, Wuhan, China, 1:1000), anti-β-actin (AC026, ABclonal, Wuhan, China, 1:20 000). The protein expression levels were normalized to corresponding β-actin or β-tubulin. ImageJ software was utilized for the quantitative analysis of Western blot results.

First, 4 µm thick TMA cores were deparaffinized and endogenous peroxidase activity was blocked using 0.3% H₂O₂ in methanol for 15 minutes. Next, slides were blocked using Ultra V Block (#TA-125-UB, Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at room temperature. Slides were then incubated with primary antibodies against C/EBPβ or C/EBPγ in PBS at 4 °C overnight (C/EBPβ 1:500 #GTX100675, GeneTex, Irvine, CA, USA; C/EBPγ 1:500 #abx103894, Abbexa Ltd., Cambridge, UK). The C/EBPβ antibody binds to the transcriptionally active C/EBPβ isoforms LAP and LAP* but not to the inactive isoform LIP. Only one isoform C/EBPδ and C/EBPγ is known to date. The next day, slides were incubated with secondary HRP-linked goat-anti-rabbit antibody (#DPVO55HRP, ImmunoLogic, Duiven, NL, USA) for 30 minutes at room temperature and stained using 3,3′Diaminobenzidine (Bright DAB #BS04-999, ImmunoLogic) with hematoxylin (1:10 in demineralized H₂O) counterstain. To assess C/EBPδ expression, the TMA and scoring published previously were employed [15 (link)].