Journal: JID Innovations

Article Title: High-salt diet aggravates skin inflammation of psoriasis-like mouse model with CCL20‒CCR6 axis further activation

doi: 10.1016/j.xjidi.2026.100451

Figure Lengend Snippet: Increasing sodium concentration upregulates Ccl20 expression in KCs through the JNK/p38–SGK1 pathway. ( a ) qPCR analysis of selected chemokines, cytokines, and Sgk1 in KCs (n = 5 for each group). (b) Immunoblot analysis of p38, phosphorylated p38, JNK, and phosphorylated JNK in KCs for different time (0 min, 30 min, 60 min), and tubulin was used as control. The relative densitometry of all bands (arbitrary unit) was normalized to the level of 0 min (n = 3 for each group). (c) qPCR analysis of Ccl20 and Sgk1 RNA expression in KCs treated with pharmacological inhibitors (n = 3 for each group). Data are shown as mean ± SEM. For a, P -values were determined by 2-tailed Student’s t -test. For c, P -values were determined by 1-way ANOVA. ∗∗ P < .01, ∗∗∗ P < .01, and ∗∗∗∗ P < .0001. KC, keratinocyte; min, minute.

Article Snippet: JNK Rabbit mAb (Cell Signaling Technology, catalog number 5292), phosphorylated JNK Rabbit mAb (Cell Signaling Technology, catalog number 4668), p38 Rabbit mAb (Cell Signaling Technology, catalog number 8690), phosphorylated p38 Rabbit mAb (Cell Signaling Technology, catalog number 4511), mouse anti-Alpha Tubulin mAb (Proteintech, catalog number HRP-66031) were used as primary antibodies, and goat antirabbit antibody (Abcam, catalog number ab6721) was used as secondary antibody.

Techniques: Concentration Assay, Expressing, Western Blot, Control, RNA Expression

Journal: Molecular cell

Article Title: Alternative splicing decouples local from global PRC2 activity.

doi: 10.1016/j.molcel.2024.02.011

Figure Lengend Snippet: Figure 2. Suz12 exon 4 skipping promotes PRC2.1 formation and dimerization (A) Schematic depiction of SUZ12 protein highlighting the relative position and amino acid (aa) sequence encoded by exon 4. GenBank NP_056170.2 (SUZ12-L); GenBank: NP_001308136.1 (SUZ12-S). (B) PRC2.2 structure surface rendering engaging a nucleosome according to Kasinath et al.23 Region encoded by exon 4 is shown in red; the SUZ12 domains C2, ZnB, and VEFS and SUZ12 Arg 196 in cyan; histone H3 Lys 27 in black; and the RBBP4 WD propeller in gray. (C) WB showing SUZ12 protein abundance in WT#2 and Dex4 cells. Dex4 samples display only the lower band corresponding to SUZ12-S. (D) Bar plot quantifying the SUZ12-L-specific and SUZ12-S-specific tryptic peptides acquired with PRM-targeted proteomics. (E) Volcano plot of SUZ12 IP-MS in Dex4 vs WT ESCs. Significant proteins (adj. p value % 0.05, log2FC > |1.5|) are shown as red points. Data were analyzed using empirical Bayes statistics on protein-wise linear models using limma in DEP (see STAR Methods). AEBP2 and JARID2 colored as in (B). (F) Stoichiometric ratio of PRC2 core, PRC2.1 and PRC2.2 interactors between WT and Dex4 cells relative to bait (SUZ12). Points indicate individual biological replicates (n = 3 clonal cell lines); error bars, sd. Data were analyzed using PSMs from proteomicslfq. Statistics: t test. (G) IP-WB of SUZ12 in WT, Dex4, or KO ESCs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies AEBP2 Cell Signaling Technology RRID:AB_2798398 BrdU-GFP BD Biosciences RRID:AB_400327 EPOP home-made Beringer et al., 20166 EZH2 BD Biosciences RRID:AB_2102429

Techniques: Sequencing, Quantitative Proteomics, Targeted Proteomics, Protein-Protein interactions

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: The IRP-IRE pull-down assays and comparison between streptavidin-agarose and -magnetic beads. ( A ) Expression of the human ferritin H gene is regulated at least three cis-acting elements. The wild type (wt) and mutant (mt) IRE (iron responsive element) RNA sequences used for pull-down probes are shown. They were 5’-end biotinylated [Btn]. ( B ) 500 μg of SW480 WCLs (60 μL in RIPA buffer) and 2 μg (equal to ~200 pmol) of biotinylated wt IRE RNA oligonucleotide were incubated in 140 μL of binding buffer A at room temperature for 1 h with constant rotation. 0–20 μL of streptavidin magnetic beads (NEB) or 20 μL of high capacity streptavidin agarose (ThermoFisher Scientific) were washed once with washing buffer, resuspended in 50 μL of binding buffer A, added to the IRE probe/WCLs mixture, and further incubated for 1 hr. The magnetic beads were collected using a magnetic stand, and agarose resins were precipitated by micro-centrifugation at 5000 rpm for 0.5 min. They were washed twice with 1 mL of washing buffer. 12 μL of 2xSDS-PAGE sample buffer was added to precipitated resins, vortexed briefly, and heated at 95 °C for 10 min. After brief spinning, the samples were loaded on 10% SDS-PAGE gel and subjected to IRP2 western blotting. WCLs of HEK293 cells transfected with empty vector, IRP2, or IRP1 expression plasmid was loaded to verify the specificity of anti-IRP2 antibody ( B – D , F ). ( C ) lanes a–e: 500 μg of SW480 WCLs (50 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 150 μL of magnetic beads binding buffer (MB, lanes a–d) or binding buffer A (lane e), followed by incubation and pull-down with 0–20 uL of pre-washed streptavidin magnetic beads (lanes a–d) or 20 μL of high capacity streptavidin agarose (lane e). lanes f, g: 500 μg of SW480 WCLs (60 μL in RIPA buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 140 μL of magnetic beads binding buffer (MB, lane f) or binding buffer A (lane g), pull-down with 20 μL of high capacity streptavidin agarose (lane g), and IRP2 western blotting. ( D ) 500 μg of SW480 WCLs (50 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 150 μL of magnetic beads binding buffer (MB, lanes a and d), binding buffer A (lanes b, e, and g), or binding buffer C (lanes c, f, and h) followed by pull-down with 20 μL or 40 μL of pre-washed streptavidin magnetic beads (lanes a–f) or 20 μL of high capacity streptavidin agarose (lane g and h), and IRP2 western blotting. ( E ) 250 μg of K562 WCLs (20 μL in IP lysis buffer) and 2 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 180 μL of binding buffer A, and pull-down with 0–30 uL of pre-washed high capacity streptavidin agarose and IRP2 western blotting. ( F ) 500 μg of SW480 WCLs (130 μL in RIPA buffer) and 2 μg of biotinylated wt or mt IRE RNA oligonucleotide were incubated in 370 μL of binding buffer A (total 500 μL) in the absence of presence of 2 μg and 8 μg of non-biotinylated wt and mt RNA oligonucleotide competitors. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and IRP2 western blotting. All experiments were repeated 2–3 times and the representative western blots are shown.

Article Snippet: Anti-IRP1 rabbit monoclonal antibody (ab126595, lot YI071316CS, abcam, Waltham, MA, USA), 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.

Techniques: Magnetic Beads, Expressing, Mutagenesis, Incubation, Binding Assay, Centrifugation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Lysis

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: Verification of the IRP-IRE pull-down assay for semi-quantitative detection of IRP2 and IRP1 binding activity. ( A ) 500 μg of SW480 WCLs (30 μL in IP lysis buffer) or ( B ) K562 WCL (40 μL in IP lysis buffer), together with 0–6 μg of biotinylated wt IRE RNA oligonucleotide were incubated in 500 μL of binding buffer A or C. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and subjected to IRP2 and IRP1 western blotting. ECL Clarity and Clarity Max (BIO-RAD) was used for IRP2 and IRP1, respectively. ( C ) 0–500 μg of K562 and SW480 WCLs were incubated with 2 μg of the biotinylated wt IRE RNA oligonucleotide in 200 μL of binding buffer A. The binding complex was pulled down with 20 μL of pre-washed high capacity streptavidin agarose and subjected to IRP2 and IRP1 western blotting. ( D ) 250 μg of WCLs (25 μL in IP lysis buffer) from K562 cells treated with 250 μM FAC or 25 μM DFO for 26 hr were incubated in 150 μL buffer C together with 4 μg of the biotinylated wt IRE probe and 20 μL of high capacity streptavidin agarose (ThermoFisher Scientific), streptavidin agarose (Invitrogen), or 30 μL of Dynabeads M-280 (Invitrogen) simultaneously. The procedures of pull-down/beads wash and western blotting for IRP2 and IRP1 are the same as other experiments. All experiments were repeated 2–3 times and the representative western blots are shown.

Article Snippet: Anti-IRP1 rabbit monoclonal antibody (ab126595, lot YI071316CS, abcam, Waltham, MA, USA), 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.

Techniques: Pull Down Assay, Binding Assay, Activity Assay, Lysis, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2

doi: 10.3390/ijms24043604

Figure Lengend Snippet: Chemicals and reagents used in this work.

Article Snippet: Anti-IRP1 rabbit monoclonal antibody (ab126595, lot YI071316CS, abcam, Waltham, MA, USA), 1000-fold dilution with 5% skim milk in Tween/TBS, 4 °C overnight.

Techniques: Magnetic Beads, Modification, Western Blot, Protease Inhibitor, Binding Assay, Lysis, Stripping Membranes, SDS Page, Plasmid Preparation