ab8227-50  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) CFIm25 overexpression increases TAB2 and TBL1XR1 mRNA expression after STM infection, assessed by RT-qPCR analysis in control and CFIm25-OE macrophages at 2 and 6 hours postinfection with STM (n = 3). Data are presented as means ± SD (n = 3); *, P < 0.05; **, P < 0.01. (B) Sequence of primers used for RT-qPCR analysis of TAB2 and TBL1XR1 APA. (C) Schematic representation of the long/total TAB2 and TBL1XR1 transcript regions targeted for RT-qPCR to assess APA and mRNA expression.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Control, Sequencing

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) Western blot analysis of CFIm25 protein in THP-1 macrophages six hours after infection with Salmonella Typhimurium (STM) compared to uninfected controls. A representative Western blot is shown on the left, and densitometric quantification of protein levels is shown on the right, normalized to GAPDH. (B-C) Flow cytometric quantification of ROS (B) and NO (C) production using DCFH-DA and DAF-FM, respectively, showing reduced microbicidal activity six hours postinfection relative to untreated macrophages. (D-E) Arginase activity, measured by urea production (D) and lactate levels (E), in macrophages six hours postinfection. All assays were compared with untreated macrophages (n = 3). Data are presented as means ± SD using three biological replicates; *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Western Blot, Infection, Activity Assay

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) Intracellular bacterial burden (CFU) in control (−) and CFIm25-overexpressing (CFIm25-OE, +) THP-1 macrophages at two and six hours postinfection. (B) Propidium iodide flow cytometric analysis of cell death in control and CFIm25-OE macrophages at two and six hours postinfection. Graphs show the percent of cells staining with propidium iodide; only dead cells take up the stain. (C) Quantification of ROS production using the DCFH-DA fluorescence assay. (D) Quantification of NO production using the DAF-FM fluorescence assay. (E) Antimicrobial activity of conditioned media from control and CFIm25-OE macrophages against STM, assessed by CFU recovery after incubation of bacteria with media. (F) Western blot analysis of CFIm25 and LL-37 protein levels in control and CFIm25-OE macrophages at two and six hours postinfection, (G) Quantitation of LL-37 levels by densitometry measurements and normalized to GAPDH. (H) Arginase activity measured by urea production in control and CFIm25-OE macrophages at two and six hours postinfection. (I) Lactate levels in control and CFIm25-OE macrophages at two and six hours postinfection were measured using a colorimetric lactate assay kit. (J) Flow cytometric analysis of M1 (CD80) and M2 (CD206) surface marker expression in control and CFIm25-OE macrophages at two and six hours postinfection. (K) Concentrations of the TNF-α, IL-12, TGF-β, and IL-10 cytokines in culture supernatants from control and CFIm25-OE macrophages at two h and six hours postinfection as measured by ELISA. Data are presented as means ± SD from three independent experiments (n = 3); *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Control, Staining, Fluorescence, Activity Assay, Incubation, Bacteria, Western Blot, Quantitation Assay, Lactate Assay, Marker, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A-B) RT-qPCR analysis of the log2 ratio of long/total TAB2 (A) and TBL1XR1 (B) mRNA isoforms in control and CFIm25-OE macrophages at two and six hours postinfection, normalized to GAPDH. (C) Western blot analysis of CFIm25, HLA-DR, CD 163, and the indicated immune signaling proteins (“P”, protein; “p”, phosphorylated form) in control and CFIm25-OE macrophages at two and six hours postinfection, with GAPDH as the loading control. (D) Quantification of changes of the indicated proteins in Western blots of samples from control and CFIm25-OE macrophages at two and six hours postinfection, with values normalized to GAPDH. (E) Representative Western blot showing nuclear and cytoplasmic distribution of NF-κB subunits in control and CFIm25-OE macrophages two and six hours postinfection, assessed by fractionation and immunoblotting. Data from all quantifications are presented as means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Quantitative RT-PCR, Control, Western Blot, Fractionation

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) CFUs recovered at six hours postinfection from Intracellular STM in CFIm25-OE macrophages transfected with control siRNA, TAB2 siRNA, or TBL1XR1 siRNA. (B) Concentrations of TNF-α, IL-12, TGF-β, and IL-10 in culture supernatants from CFIm25-OE macrophages expressing control, TAB2, or TBL1XR1 siRNAs, harvested at six hours postinfection, and measured by ELISA. (C-F) ROS (C), NO (D), arginase activity (E), and lactate (F) levels at six hours postinfection in CFIm25-OE macrophages treated with control, TAB2, or TBL1XR1 siRNA. (G) Flow cytometric analysis of CD80 and CD206 expression at six hours postinfection in CFIm25-OE macrophages treated with control, TAB2, or TBL1XR1 siRNA. (H) Western blot analysis of the indicated NF-κB and MAPK signaling proteins at six hours postinfection in CFIm25-OE macrophages treated with control, TAB2, or TBL1XR1 siRNA, with densitometric quantification from three independent experiments (“P”, protein; “p”, phosphorylated form). Data are presented as means ± SD (n = 3); *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Transfection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot