Igg isotype control

ChIP‐seq PCR was performed using the ChIP assay kit (Cell signaling technology, Denver, CO) as described previously (Sontake et al, 2018). Briefly, lung‐resident fibroblasts were purified from lung stromal culture of CCSP/TGFα mice on Dox‐containing food for 8 weeks. Cells were cross‐linked with 1% formaldehyde, and immunoprecipitation was performed with anti‐WT1 (catalog 12609‐1‐AP, Proteintech, Rosemont, IL) or isotype control IgG (Cell Signaling Technology, Denver, CO) at 4°C as described in the manufacturer's protocol. DNA was purified, and ChIP‐qPCR was performed using the CFX384 Touch Real‐Time PCR detection system and SYBR select master mix (Bio‐Rad, Hercules, and CA).

Internalization assays were performed essentially as described38 (link), with slight modification. For VEGFR1 internalization, HUVEC were washed five times with pre-chilled PBS supplemented with Ca+2 and Mg+2 (PBS+) on ice at 4 °C. Following washing, HUVEC were incubated with ice-cold blocking/internalization solution EBM-2 supplemented with 0.5% w/v BSA for 30 min on ice at 4 °C to prevent internalization. Alexa-fluor-488 conjugated primary antibody against VEGFR1 (Abcam Cat # 195253) or IgG isotype control (Cell Signaling Technologies Cat t# 4340) was added (1:50) to pre-chilled blocking solution incubated with HUVEC for 2 h at 4 °C to label cell surface proteins. After 2 h, cells were washed five times in ice cold PBS+ before adding pre-warmed (37 °C) internalization medium to each plate and incubating at 37 °C for 45 min to activate endocytosis. When striping of the cell surface was performed, HUVEC were incubated with 0.5 NaCl/0.2 M acetic acid for 4 min after internalization but before fixation to remove antibody from cell surface receptors.

Bone marrow-derived neutrophil cells extracted from Smad3-WT mice (pooled from four mice /group) were treated with 10% LLC-CM or normal media (control) for 2 h, then processed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s instruction^{58 (link)}. Antibodies against Smad3 and IgG isotype control (Cell Signaling Technology) were used for immunoprecipitation. Smad3 immuno-enriched DNA (input and Smad3-IP) were sequenced using the Illumina HiSeq platform (PE150bp, 40 M raw read), mapped against GRCm38 (mm10) Mus musculus genome using Bowtie2, and peaks identified using model-based analysis for ChIP-Seq (MACS) with default parameters. Normalized Smad3 binding regions were visualized by EaSeq^{59 (link)}, and annotated by the Genomic Regions Enrichment of Annotations Tool (GREAT) for DAVID-GO enrichment analysis. Motif analysis was performed in Trawler (trawler.erc.monash.edu.au) with default parameters.

Co-immunoprecipitation was performed using the IP/CoIP kit (Absin, Shanghai, China) according to the manufacturer’s instructions. Primary antibodies against PKM2 and STAT1 were purchased from Cell Signaling Technology (Boston, MA, USA) and Abcam (Cambridge, CB2 0AX, UK), respectively. The IgG isotype control was purchased from Cell Signaling Technology (Boston, MA, USA). A secondary antibody specific to the IgG light chain was purchased from ABclonal (Wuhan, China).

Cells were lysed in 100 μl of Frackelton lysis buffer (10 mM Tris [pH 7.4], 50 mM NaCl, 30 mM Na₄P₂O₇, 50 mM NaF, 2 mM EDTA, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and 1X protease inhibitor cocktail). Cells were incubated on a rotating wheel at 4°C for 30 min and subsequently centrifuged at 20,000 × g at 4°C for 30 min. The supernatant was transferred to a new tube and 10 μl (10% of the lysate used for the immunoprecipitations [IPs]) was collected as input. 300 μg of lysates were incubated overnight at 4°C on a rotating wheel with an IgG Isotype Control (Cell Signaling Technology, 1:300), anti-Ollas (Novus, 1:100), or anti-IRF1 antibody (Cell Signaling Technology, 1:100). The next day, magnetic beads (Pierce Protein A/G Magnetic Beads, Thermo Fisher Scientific, 88803) used for anti-IRF1 antibody IPs, or Protein G Sepharose beads (Protein G Sepharose 4 Fast Flow, Sigma-Aldrich, GE17-0618-01) used for anti-Ollas antibody IPs, were blocked by rotation in 3% BSA in Frackelton Buffer for 1 hr at 4°C. 25 μl of beads were added to 300 μg of lysates and rotated for 2 hr at 4°C. Then, the beads were washed five times with 1 ml of RIPA buffer, supplemented with 300 mM NaCl. Proteins were eluted by boiling in 2X disruption buffer (2.1 M urea, 667 mM β-mercaptoethanol, and 1.4% SDS) for 5 min at 95°C.

KGN cells were plated at a density of 2 × 10⁵ cells and cultured for 24 hours with each treatment condition. After collection of cells, ChIP-qPCR was performed by using ChIP-IT Express Chromatin Immunoprecipitation Kits (Active Motif, CA, USA) according to the manufacturer’s protocol. The antibodies used included anti-AhR (Abcam, Ab2769) and an IgG isotype control (Cell Signaling Technology, 3900). The Glut4 primer sequences were 5′-ATGACCTCTGGTCACCAAACTG-3′ (sense) and 5′-GATCAGTCAGAAGCATAGGTGAC-3′ (antisense). Real-time PCR amplification was carried out and amplification of target gene is shown as fold enrichment compared to that of irrelevant antibody controls. The results were from 3 independent experiments followed by normalization to input signals and showed as mean ± SEM.