Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Top: schematic of the K562 doxycycline-inducible system from Hansen et al. (2022a), in which FOXA1 and HNF4A were induced individually or together in cells lacking endogenous expression of either factor. Bottom: representative CUT&Tag tracks at one peak from each category, showing FOXA1 antibody signal (blue) and HNF4A antibody signal (orange) across the three induction conditions. Co-bound sites (peaks present in both dual-induction antibody tracks; 50% reciprocal overlap on narrowPeak intervals) were classified by their dependence on single-TF expression. FOXA1-enabled (FE, n = 1,510): bound by FOXA1 in the FOXA1-only condition. HNF4A-enabled (HE, n = 2,727): bound by HNF4A in the HNF4A-only condition. Cooperative (CB, n = 1,824): bound by neither factor in either single-TF condition. Redundant (n = 1,875): bound by both factors in their respective single-TF conditions. (b) Per-peak baseline (uninduced) ATAC-seq signal by category. ATAC-seq from GSE182188, same K562 doxycycline-inducible system. (c) Change in per-peak ATAC-seq signal upon dual induction (ΔATAC = induced − uninduced). Dashed line: no change. In b and c, box plots show median (centre line), interquartile range (box), and 1.5×IQR whiskers; violins show the underlying data distribution. Brackets show two-sided Mann–Whitney U tests comparing Cooperative against each other category (****p < 0.0001). (d) Log₂ fold-enrichment of each site category over genome-wide background across seven summary chromatin states consolidated from the Broad 15-state ChromHMM K562 segmentation (wgEncodeBroadHmm). Fold enrichment = (fraction of category overlapping state) / (genomic fraction of state). Cell values are fold enrichments; colour, log₂(fold enrichment). (e) Mean MNase-seq nucleosome occupancy in a ±1 kb window centred on each peak summit, by category. MNase-seq from Mieczkowski et al. 2016 (GEO GSM2083140) . Lines show category means; shaded bands show ±SEM. Sites with usable bigWig coverage (≥50% non-NaN bins): FE n = 1,420; HE n = 2,560; CB n = 1,781; RD n = 1,822. Signal binned at 10 bp and Gaussian-smoothed (σ = 20 bp).

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Expressing, MANN-WHITNEY, Genome Wide

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Dual-head binding CNN architecture. One-hot encoded 1,001 bp sequences (summit ± 500 bp) pass through three convolutional blocks (64/128/128 filters; kernel sizes 19/11/7; each: Conv → BatchNorm → ReLU → MaxPool(4) → Dropout 0.25), global average pooling, and two independent task-specific MLP heads with sigmoid output. Training: 138,489 sequences (peaks from all four categories vs. cis-regulatory negatives from uninduced K562 ATAC-seq); chromosome-based splits (test: chr1, chr8, chr9; validation: chr2, chr3). (b) ROC (left) and precision-recall (right) on the held-out test set. FOXA1 head: AUROC = 0.868, AUPRC = 0.792; HNF4A head: AUROC = 0.878, AUPRC = 0.731. (c) DeepLIFT attribution heatmaps by category, after SVA filtering of the HNF4A-Enabled set (see Supplementary Fig. 4): FOXA1-Enabled (n = 1,507), HNF4A-Enabled (n = 2,105; 622 SVA-overlapping sites removed), Co-Bound (n = 1,775), Redundant (n = 1,865). Left: FOXA1 head importance (blue); right: HNF4A head importance (orange). Each row is one site; rows are sorted by position of peak attribution. Each head’s attribution is strongest at its single-TF-enabled category; both heads contribute at Co-Bound sites. (d) Total CNN head attribution within ±250 bp of the peak summit by category. FOXA1 head (left) is most active at FOXA1-Enabled sites (median 3.83 vs. 2.44 at HNF4A-Enabled); HNF4A head (right) is most active at HNF4A-Enabled sites (median 4.49 vs. 2.50 at FOXA1-Enabled). The per-site cognate-head attribution fraction (cognate-head attribution / total attribution) is higher at HNF4A-Enabled than FOXA1-Enabled sites (63.7% vs. 58.4%; two-sided Mann–Whitney p = 2.3 × 10⁻²⁴). (e) FIMO-based motif counts within ±250 bp of the peak summit (FIMO p < 10⁻³; JASPAR MA0148.1, MA0114.2). FOXA1-Enabled sites carry more FOXA1 motifs (median 3) than HNF4A motifs (median 2); HNF4A-Enabled sites show the reverse (median 4 vs. 2). The cognate-motif fraction is correspondingly higher at HNF4A-Enabled sites (71.4% vs. 57.1%; p = 7.8 × 10⁻⁸⁷). In d and e, box plots show median (centre line), interquartile range (box), and 1.5×IQR whiskers; violins show the underlying data distribution.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Binding Assay, Biomarker Discovery, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: Spacing: centre-to-centre between lowest-p FOXA1 (MA0148.1) and HNF4A (MA0114.2) motifs per peak, summit ± 500 bp, max 500 bp inter-motif (best-score pairing; FIMO p < 10⁻³). (a) Kernel density of per-peak motif spacing by category; lines mark medians. Cooperative is shortest (146 bp) vs FOXA1-enabled (180), HNF4A-enabled (182; SVA-filtered), Redundant (197); n = peaks with both motifs per category. (b) Per-bin log₂(observed/expected) in 5 bp bins against a 1,000-permutation per-peak null (motif positions shuffled within the 1,001 bp window). Dark red, FDR-enriched (BH q < 0.05); dark blue, depleted; pale, n.s. Cooperative shows 12 enriched bins at 15–60 bp; FOXA1-enabled and Redundant show 0; HNF4A-enabled shows 1 (Suppl. Fig. 6 for unfiltered). (c) Same pipeline at endogenously co-bound sites. Top: K562 Cooperative, replotted from (b). Second: HepG2 (FOXA1–HNF4A ChIP-seq, GSE104247; 9,373 motif pairs), 14 enriched bins. Third: HDMA fetal hepatocyte caCREs (Liu et al. 2026, Nature; 44,165 peaks in clusters LI_1/3/4/6 from 29,926 cells, PCW15–22; 24,327 motif pairs), 10 enriched bins concentrated at 15–60 bp. Bottom: HDMA fetal brain caCREs (BR_0–BR_17; 74,035 peaks, 14,015 motif pairs), 0 enriched bins.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Single-site Pioneer-seq library design. A single FOXA1 binding site (blue; TGTTTACTTTG, JASPAR MA0148.1) or a single HNF4A binding site (orange; GAGTCCAAAGTCCAG, JASPAR MA0114.2) was placed at each of 182 centre positions (−85 to +96 bp relative to the dyad) on three reconstituted nucleosomal templates: Widom-601, 5S rDNA, and mouse mammary tumor virus (MMTV)-A. A paired nonspecific control sequence (a partial ETS motif; ACCGGAAGTG, JASPAR MA0098.3) was placed at matched positions on the same templates. Each row of the schematic represents one library member. (b) Relative shift (RS) as a function of binding-site centre position relative to the nucleosome dyad. Top row: FOXA1 (blue) and the paired nonspecific control (grey). Bottom row: HNF4A (orange) and the paired nonspecific control. Points show the mean of n = 3 biological replicates; vertical error bars, SEM. Vertical dashed lines mark the dyad (position 0) and the canonical nucleosome boundaries (±73 bp). RS is defined as −log₂((T / T_NS) / (N / N_NS)), where T and T_NS are read counts of the test and paired nonspecific-control nucleosomes in the unshifted band of the TF-treated lane, and N and N_NS are the corresponding counts in the no-TF (null) lane (Methods). (c) Binding ability per template, defined as the mean excess RS over the nonspecific control, ⟨RS_TF − RS_NS⟩, averaged across all 182 positions. Bars show the mean; error bars, SEM propagated from per-position SEMs. p-values, one-sided paired Wilcoxon signed-rank test (alternative: FOXA1 > HNF4A; 182 paired positions per template); the directional hypothesis was prespecified from cellular observations (Hansen et al., 2022a) of FOXA1’s lower per-motif binding requirement.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Binding Assay, Virus, Control, Sequencing

Journal: bioRxiv

Article Title: Cooperative FOXA1–HNF4A binding emerges from motif spacing and nucleosome architecture

doi: 10.64898/2026.05.27.728252

Figure Lengend Snippet: (a) Cobinding Pioneer-seq library design. On each of three nucleosomal templates (Widom-601, 5S rDNA, mouse mammary tumor virus (MMTV)-A; light-to-dark grey shading), a FOXA1 site (blue) and an HNF4A site (orange) were placed adjacently with a fixed 5 bp gap between the two sites, at each of 77 outermost-site positions (bp 21–97 from the dyad). Each row in the schematic represents one library member. (b) Pioneer-seq relative shift (RS) as a function of the outermost site’s distance from the dyad. Green: FOXA1–HNF4A composite (TGTTTACTTTG–N₅–GAGTCCAAAGTCCAG; JASPAR MA0148.1 + MA0114.2). Blue: FOXA1 alone (MA0148.1). Orange: HNF4A alone (MA0114.2). Grey: paired nonspecific control (ACCGGAAGTG; JASPAR MA0098.3). Per-position values are the mean of n = 3 biological replicates with SEM error bars. Vertical dashed line marks the canonical nucleosome edge (bp 73). Large green dots mark positions where the cobinding signal exceeds the sum of single-TF signals on the linear scale (2^FH > 2^F + 2^H; paired z-test with delta-method error propagation; Bonferroni-corrected across the 77 positions per template, α = 0.05). Cartoons at right depict the four binding conditions, colour-matched to the trace lines. (c) Genomic-nucleosome library. Each row represents one of n = 179 nucleosomes selected from K562 Cooperative-category peaks containing exactly one FOXA1 motif (blue) and exactly one HNF4A motif (orange) (FIMO p < 10⁻³, JASPAR MA0148.4 for FOXA1 and MA0114.4 for HNF4A; nucleosome dyads inferred by DANPOS from K562 MNase-seq (Mieczkowski et al. 2016) at occupancy score ≥ 0.7; Methods). (d) Cobinding RS on the genomic-nucleosome library versus the FOXA1 motif’s distance from the inferred dyad (left) and the HNF4A motif’s distance from the inferred dyad (right). Points, individual nucleosomes; line, ordinary least squares fit; grey band, 95% CI. In-panel: Pearson r, two-sided p, and n. Banner: Δr = r_FOXA1 − r_HNF4A and two-sided Fisher z-test comparing the two Pearson correlations.

Article Snippet: Recombinant human full-length FOXA1 (Origene TP306045) and HNF4A (Origene TP317863) were used for binding reactions.

Techniques: Virus, Control, Binding Assay

Journal: PLOS Pathogens

Article Title: Equine infectious anemia virus blocks interferon responses through Rev-mediated activation of the stress granule-PKR-eIF2α pathway

doi: 10.1371/journal.ppat.1014262

Figure Lengend Snippet: ( A-C ) mRNA levels of MDA5, IFITM3, and ISG15 in uninfected and EIAV-infected eMDMs. eMDMs were either mock-infected or infected with EIAV (MOI = 1) for 12 or 24 h, and then treated with either 100 U/mL IFN-α or mock-treated with phosphate-buffered saline (PBS) for an additional 12 hours. Cellular RNA was extracted, and the levels of MDA5 (A) , IFITM3 (B) , and ISG15 (C) mRNA were determined using RT-qPCR. β-actin mRNA quantification from the same samples was used for normalization. Data represent means ± SD of three independent experiments. (D) Protein levels of MDA5, IFITM3, and ISG15 in uninfected and EIAV-infected eMDMs. Western blot analysis was performed to examine the protein expression of STAT1, p-STAT1, MDA5, IFITM3, and ISG15 in the samples corresponding to Fig 1A. β-actin was used as a loading control, and EIAV infection was verified using an anti-P26 antibody. The intensities of the protein bands were analyzed using the Odyssey Imaging System to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated mock cells (24 h) as the control group, which was set to 1.

Article Snippet: Equine IFN-α protein (RP0142E), equine IFN-β protein (RP0935E-005) and human IFN-α protein (RP1628H) were purchased from Kingfisher Biotech.

Techniques: Infection, Saline, Quantitative RT-PCR, Western Blot, Expressing, Control, Imaging

Journal: PLOS Pathogens

Article Title: Equine infectious anemia virus blocks interferon responses through Rev-mediated activation of the stress granule-PKR-eIF2α pathway

doi: 10.1371/journal.ppat.1014262

Figure Lengend Snippet: EIAV suppresses ISG protein production through the PKR-eIF2α pathway. (A) Assessment of host translation activity in EIAV-infected eMDMs using a ribopuromycylation assay. eMDMs were infected with EIAV at an MOI of 1 for 12, 24, or 36 hours. Prior to harvest, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins. Whole cell lysates were analyzed using western blotting with the indicated antibodies. (B) Dose-dependent activation of the PKR-eIF2α pathway resulting from EIAV proviral DNA expression. HEK293T cells were seeded into 6-well plates and transfected with increasing concentrations (250, 500, and 1000 ng) of pCMV3-8 or empty vector. At 24 hpt, whole cell lysates were analyzed using western blotting with the indicated antibodies. (C) Treatment with the PKR inhibitor C16 blocks activation of the PKR-eIF2α pathway resulting from EIAV proviral DNA expression. HEK293T cells were transfected with pCMV3-8 (1 μg) or empty vector. At 16 hpt, cells were treated with 1 μM C16 or DMSO for an additional 12 h before harvest. Prior to harvest, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins. Whole cell lysates were analyzed using western blotting with the indicated antibodies. (D) Treatment with the PKR inhibitor C16 restores expression of ISG proteins in EIAV-infected cells. eMDMs were infected with EIAV (MOI = 1) for 10 h, then treated with 1 μM C16 or DMSO for 2 h prior to stimulation with 100 U/mL IFN-α or PBS for 12 h. Prior to harvest, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins. Whole cell lysates were analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated mock cells that were treated with DMSO. (E) C16 treatment reduces viral replication. Cell culture supernatants from samples corresponding to those in Fig 2D were collected. Viral RNA was extracted from the supernatants and quantified using qPCR to determine viral copy numbers. Data represent means ± SD of three independent experiments.

Article Snippet: Equine IFN-α protein (RP0142E), equine IFN-β protein (RP0935E-005) and human IFN-α protein (RP1628H) were purchased from Kingfisher Biotech.

Techniques: Activity Assay, Infection, Synthesized, Western Blot, Activation Assay, Expressing, Transfection, Plasmid Preparation, Cell Culture

Journal: PLOS Pathogens

Article Title: Equine infectious anemia virus blocks interferon responses through Rev-mediated activation of the stress granule-PKR-eIF2α pathway

doi: 10.1371/journal.ppat.1014262

Figure Lengend Snippet: EIAV Rev activates the PKR-eIF2α pathway to suppress induction of ISG protein. (A) Screening of viral proteins for inducing phosphorylation of PKR and eIF2α. HEK293T cells were transfected with HA-tagged Gag, Env, Tat, Rev, S2, Mat, Grev, dUTPase, S4 or with empty vector. At 24 hpt, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins prior to harvest. Protein expression was analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to control cells. (B) Overexpression of Rev inhibits expression of interferon-stimulated proteins. HEK293T cells were transfected with pcDNA 3.1-Rev-HA or empty vector (500 ng). After 20 h, cells were stimulated with 100 U/mL IFN-α or PBS for 12 h. Prior to harvest, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins. Protein expression was analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated cells transfected with empty vector. (C-E) Overexpression of Rev does not affect ISG mRNA levels. RT-qPCR analysis was performed to examine the levels of MDA5 (C), IFITM3 (D), and ISG15 (E) mRNA in the samples corresponding to those in Fig 3B. Data represent means ± SD of three independent experiments. (F) Treatment with the PKR inhibitor C16 restores expression of ISG protein suppressed by Rev overexpression. HEK293T cells were transfected with Rev-expressing plasmid for 16 hours and then treated with the PKR-specific inhibitor C16 (0.8 μM) for 2 hours prior to IFN-α stimulation (100 IU/mL) for 12 hours. Prior to harvest, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins. Protein expression was analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated cells transfected with empty vector (DMSO control).

Article Snippet: Equine IFN-α protein (RP0142E), equine IFN-β protein (RP0935E-005) and human IFN-α protein (RP1628H) were purchased from Kingfisher Biotech.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Synthesized, Expressing, Western Blot, Control, Over Expression, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Equine infectious anemia virus blocks interferon responses through Rev-mediated activation of the stress granule-PKR-eIF2α pathway

doi: 10.1371/journal.ppat.1014262

Figure Lengend Snippet: EIAV Rev activates the PKR-eIF2α pathway and suppresses induction of ISG protein in a G3BP1-dependent manner. (A) G3BP1 interacts with Rev. HEK293T cells were co-transfected with pcDNA3.1-Rev-HA (500 ng) and either an empty vector or pCMV-G3BP1-Flag (500 ng). At 24 hpt, cell lysates were prepared and treated with or without RNase A (100 μg/mL) before immunoprecipitation with anti-Flag M2 beads. Immunoprecipitates were then analyzed using western blotting with anti-HA, anti-Flag, and anti-β-actin antibodies. For reciprocal Co-IP, HEK293T cells were co-transfected with pCMV-G3BP1-Flag and either an empty vector or pcDNA3.1-Rev-HA. After 24 hpt, cell lysates were prepared and treated with or without RNase A before immunoprecipitation with anti-HA beads. The precipitated proteins were then analyzed using western blotting with the indicated antibodies. (B) Schematic diagram of wild-type G3BP1 and structures of respective mutants. (C) Rev Interacts with the NTF2L domain of G3BP1. HEK293T cells were co-transfected with pcDNA3.1-Rev-HA and either an empty vector, wild-type G3BP1, or truncated G3BP1-Flag. At 24 hpt, cells were harvested, immunoprecipitated with anti-Flag antibody, and further assessed using western blotting with the indicated antibodies. (D) Knockout of G3BP1 restores Rev-mediated suppression of ISG proteins. G3BP1 knockout HEK293T cells and wild-type controls were transfected with pcDNA3.1-Rev-HA or empty vector (500 ng) for 20 hours. Cells were then stimulated with 100 U/mL IFN-α for an additional 12 hours. Protein expression was analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated wild-type cells transfected with empty vector. (E) Knockdown of G3BP1 restores expression of ISG proteins in EIAV-infected cells. eMDMs were transfected with G3BP1-specific siRNA for 24 hours, followed by infection with EIAV (MOI = 1) for 12 hours. Prior to harvest, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins. Cells were then stimulated with 100 U/mL IFN-α for an additional 12 hours. Protein expression was analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated mock cells. (F) G3BP1 depletion inhibits viral replication. Cell culture supernatants from samples corresponding to Fig 5E were collected. Viral RNA was extracted from the supernatants and quantified with qPCR to determine viral copy numbers. Data represent means ± SD of three independent experiments.

Article Snippet: Equine IFN-α protein (RP0142E), equine IFN-β protein (RP0935E-005) and human IFN-α protein (RP1628H) were purchased from Kingfisher Biotech.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Knock-Out, Expressing, Knockdown, Infection, Synthesized, Cell Culture

Journal: PLOS Pathogens

Article Title: Equine infectious anemia virus blocks interferon responses through Rev-mediated activation of the stress granule-PKR-eIF2α pathway

doi: 10.1371/journal.ppat.1014262

Figure Lengend Snippet: (A) Schematic diagram of wild-type EIAV Rev and structures of respective mutants. (B) Immunoblot analysis of ISG protein expression in HEK293T cells transfected with EIAV Rev mutants. HEK293T cells were transfected with plasmids encoding Rev-WT or with one of the five Rev mutants (500 ng) as described in Fig 6A. At 20 hpt, cells were stimulated with 100 U/mL IFN-α for an additional 12 hours. Cell lysates were then prepared and subjected to western blot analysis using the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to IFN-α-treated cells transfected with wild-type Rev. (C) Immunofluorescence analysis of SG formation induced by Rev mutants. HeLa cells were transfected with plasmids encoding wild-type Rev or one of the various Rev mutants. At 24 hpt, cells were fixed and stained with the anti-G3BP1 antibody (green), anti-HA antibody (red, for Rev detection), and DAPI (blue) for nuclear staining. Scale bars: 10 µm. Shown is an intensity profile of the linear ROI across the HeLa co-stained with G3BP1 and Rev. (D) Quantification of SG-positive cells in Fig 6C. Data represent means ± SD of three independent experiments. (E) Immunoblot analysis of PKR/eIF2α phosphorylation and puromycin incorporation in HEK293T cells transfected with EIAV Rev mutants. HEK293T cells were transfected with plasmids encoding Rev-WT or with one of the five Rev mutants (500 ng) as described in Fig 6A. At 24 hpt, cells were pulsed with 5 µg/mL puromycin for 30 min to label newly synthesized proteins prior to harvest. Protein expression was analyzed using western blotting with the indicated antibodies. The intensities of the protein bands were analyzed to calculate values relative to those of β-actin. Results were normalized to cells transfected with wild-type Rev. (F) Co-IP analysis of G3BP1 and Rev mutants. HEK293T cells were co-transfected with pCMV-G3BP1-Flag and an empty vector, or wild-type Rev or one of the Rev mutants. At 24 hpt, cells were harvested, immunoprecipitated with anti-HA antibody, and further assessed using western blotting with the indicated antibodies.

Article Snippet: Equine IFN-α protein (RP0142E), equine IFN-β protein (RP0935E-005) and human IFN-α protein (RP1628H) were purchased from Kingfisher Biotech.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Staining, Phospho-proteomics, Synthesized, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation