Journal: iScience

Article Title: Unveiling critical signaling pathways in the murine salivary gland and the role of midkine

doi: 10.1016/j.isci.2026.114852

Figure Lengend Snippet: Midkine associated signaling in the murine SMG (A) Heatmap visualization of the MDK signaling network across various cell populations in the mouse SMG. (B) Violin plot showing expression levels of ligands and receptors associated with MDK signaling in the different cell populations of the mouse SMG at E16. (C) Chord diagram depicting outgoing communication probability of ligand-receptor pairs contributing to MDK signaling from mesenchyme (upper panel) and MECs (lower panel), based on results from panel A. (D) Representative immunofluorescence images of mouse SMGs co-stained with Mdk and the acinar cell marker Nkcc1 or the ductal marker K19 at E13, E14, and E16. Scale bars, 20 μm.

Article Snippet: Primary antibodies used at the indicated dilutions include Midkine (R&D Systems, 1:100), Midkine (ProteinTech, 1:100), K14 (Rizzo et al., 2016, 1:100), Sma (Sigma 1A4, 1:200), Nkcc1 (Santa Cruz Biotechnology, 1:100), ΔNp63 (Cell Signaling Technology, 1:50), ΔNp63 ( ,1:50) Mist1 (Abcam, 1:100), Aqp5 (Alomone Labs, 1:100), Tubb3 (R&D Systems, 1:100), K7 (Abcam, 1:50), K5 (gift from Dr. Julie Segre, 1:100), Troma-III/K19 (Development Studies Hybridoma Bank, 1:50), and Rock1 (Cell Signaling Technology, 1:25).

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: iScience

Article Title: Unveiling critical signaling pathways in the murine salivary gland and the role of midkine

doi: 10.1016/j.isci.2026.114852

Figure Lengend Snippet: Branching morphogenesis of embryonic E13.5 SMGs treated with midkine inhibitors is reduced in ex vivo culture (A) Upper panel are light micrographs of wild type C57BL/6J SMGs harvested at E13.5 and cultured for 72 h in the presence of midkine inhibitor (iMDK) or DMSO control. Lower panel shows whole-mount immunofluorescence staining of DMSO control and iMDK treated glands shows expression of Keratin 5 (K5) and neuronal tubulin expressing nerves (Tubb3). Scale bars, 100 μm, ( n = 3). (B) Spooner’s ratio quantification of the number of end buds (expressed as a ratio of the number of end buds at 72 h/number at 1 h) of DMSO control ( n = 10) and iMDK treated glands ( n = 10). (C) Immunofluorescence staining of paraffin embedded DMSO control and iMDK treated glands reveal decreased expression of ΔNp63 and reduced expression of K14 expressing basal stem/progenitor cells. Alterations to the acinar cell differentiation program were also observed in the iMDK treated glands as evident by reduced expression of Mist1, Nkcc1, and Aqp5. Ductal cell differentiation was also altered as depicted by decreased K7 expression in the iMDK treated glands compared to DMSO controls. Scale bars, 20 μm. Lower panel shows quantification of the p63 + K14 + /p63 + basal cells, p63 + Sma + /p63 + MECs, and Nkcc1 + Mist1 + /Nkcc1 + acinar cell population as mean ± SD ( n = 3). (D) Quantitative RT-PCR validation of select genes in the DMSO control and iMDK treated glands. Values were normalized to the housekeeping gene Hprt . Data are represented as means ± SD ( n = 3). ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies used at the indicated dilutions include Midkine (R&D Systems, 1:100), Midkine (ProteinTech, 1:100), K14 (Rizzo et al., 2016, 1:100), Sma (Sigma 1A4, 1:200), Nkcc1 (Santa Cruz Biotechnology, 1:100), ΔNp63 (Cell Signaling Technology, 1:50), ΔNp63 ( ,1:50) Mist1 (Abcam, 1:100), Aqp5 (Alomone Labs, 1:100), Tubb3 (R&D Systems, 1:100), K7 (Abcam, 1:50), K5 (gift from Dr. Julie Segre, 1:100), Troma-III/K19 (Development Studies Hybridoma Bank, 1:50), and Rock1 (Cell Signaling Technology, 1:25).

Techniques: Ex Vivo, Cell Culture, Control, Immunofluorescence, Staining, Expressing, Cell Differentiation, Quantitative RT-PCR, Biomarker Discovery

Journal: The Journal of Clinical Investigation

Article Title: Chloride homeostasis dysfunction drives hyperactivation of corticotropin-releasing factor-expressing neurons in the amygdala in stress-induced hypertension

doi: 10.1172/JCI195536

Figure Lengend Snippet: ( A – C ) Representative immunoblots and quantification of membrane NKCC1 protein in the CeA ( A ), frontal cortex ( B ) and RVLM ( C ) of WKY rats, CUMS WKY rats, BHRs and CUMS BHRs ( n = 6 rats in each group). ( D – F ) Representative immunoblots and quantification of membrane KCC2 protein in the CeA ( D ), frontal cortex ( E ) and RVLM ( F ) of WKY rats, CUMS WKY rats, BHRs and CUMS BHRs ( n = 6 rats in each group). Data are expressed as means ± SEM. ** P < 0.01. One-way ANOVA followed by Tukey’s post hoc test. ( G ) Representative immunofluorescent images show CRF-positive neurons (green), NKCC1-positive neurons (red), and NKCC1 and CRF double-labeled neurons in the CeA of BHR and CUMS BHR ( n = 5 rats in each group). ( H and I ) The percentage of CRF ( H ) or NKCC1 ( I ) positive neurons in the CeA of BHR and CUMS BHR. ( J ) The percentage of CRF and NKCC1 double positive neurons in the CeA of BHR and CUMS BHR. CeA, central nucleus of amygdala. RVLM, rostral ventrolateral medulla. Data are expressed as means ± SEM. *** P < 0.001. Two-tailed Student’s t -test.

Article Snippet: Subsequently, the sections were incubated with a mouse anti-delta-FosB antibody (1:50, #sc-398595), a rabbit anti-CRF antibody (1:100, #A1122, Abclonal) or a rabbit anti-NKCC1 antibody (1:100, #13884, Proteintech) overnight at 4°C.

Techniques: Western Blot, Membrane, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Functional differences in electrolyte transport between the mouse proximal and distal trachea

doi: 10.64898/2026.02.26.708204

Figure Lengend Snippet: (A) representative frontal section of the trachea from an adult wild-type (WT) mouse, showing immunostaining for the Sodium-Potassium-Chloride cotransporter 1 (NKCC1) in the surface epithelium (SAE). (B) Higher magnification of SMGs, (C) proximal, (D) medial and (E) distal regions of the tracheal and (F) and main bronchi SAE for the WT mouse. The right column (B’-F’) shows higher-magnification views of the regions indicated by the insets in panels B–F. White dashed lines outline the submucosal glands (SMGs), yellow dashed lines delineate the basal lamina of the SAE, and light blue dashed lines indicates cricoid cartilage (Cr). Nuclei are counterstained with DAPI. Scale bars: 200 µm (A), 50 µm (B–F), and 10 µm (B’–F’). Representative images of wild type (n=6). Thyroid Gland (Thy). (G) forskolin-induced I eq , (H) succinate-induced I eq , (I) carbachol-induced I eq and (J) UTP-induced I eq , wild type Slc12a2 +/+ or Slc12a2 -/- tissues; n=5 for each group.; t-student paired-test for proximal (blue dots) versus distal (red dots) on each group and t-student unpaired-test in comparisons between different groups.

Article Snippet: 8-week-old WT mice were incubated with anti-NKCC1 antibody (D2O8R XP; Cell Signaling, cat# 85403; 1:100), followed by Alexa Fluor 488 goat anti-rabbit IgG (1:2000; Invitrogen, cat# A-11008).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Functional differences in electrolyte transport between the mouse proximal and distal trachea

doi: 10.64898/2026.02.26.708204

Figure Lengend Snippet: (A) representative frontal section of the trachea from an adult wild-type (WT) mouse, showing immunostaining for the Sodium-Potassium-Chloride cotransporter 1 (NKCC1) in the surface airway epithelium (SAE). (B) Higher magnification of SMGs, (C) proximal and (D) distal regions of the tracheal SAE for the wild type (A-D) and Slc12a2 -/- (B’-D’). Representative images of NKCC1+ cells in the (E) main bronchi, (F) bronchi and (G) terminal bronchi, for wild type (E-G) and Slc12a2 -/- (E’-G’). White dashed lines outline the submucosal glands (SMGs), and yellow dashed lines delineate the basal lamina of the SAE. Nuclei are counterstained with propidium iodide. Scale bars: 100 µm (A), 20 µm (B–G). Representative images of wild type (n=4) and Slc12a2 -/- (n=3).

Article Snippet: 8-week-old WT mice were incubated with anti-NKCC1 antibody (D2O8R XP; Cell Signaling, cat# 85403; 1:100), followed by Alexa Fluor 488 goat anti-rabbit IgG (1:2000; Invitrogen, cat# A-11008).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Functional differences in electrolyte transport between the mouse proximal and distal trachea

doi: 10.64898/2026.02.26.708204

Figure Lengend Snippet: (A-C) Representative images from sections of the tracheal surface epithelium from an adult Ascl3 PGA-GCE ; Rosa26 tdTOMATO mouse, showing immunostaining for the Sodium-Potassium-Chloride cotransporter 1 (NKCC1 high )+ cells (green) and ASCL3+ cells (Magenta). (A, B) Colocalization of NKCC1 and ASCL3 is observed in some cells (Weighted colocalization coefficients: 0.78118 and 0.99441, respectively), whereas (C) shows a lack of colocalization. Yellow dashed lines delineate the basal lamina of the tracheal epithelium, and white dashed lines outline the submucosal glands (SMGs). Nuclei are counterstained with DAPI. Scale bars 10 µm, n=3.

Article Snippet: 8-week-old WT mice were incubated with anti-NKCC1 antibody (D2O8R XP; Cell Signaling, cat# 85403; 1:100), followed by Alexa Fluor 488 goat anti-rabbit IgG (1:2000; Invitrogen, cat# A-11008).

Techniques: Immunostaining

Journal: Acta Neuropathologica Communications

Article Title: Temporal and cell-type specific SPAK-NKCC1 disruption following severe TBI in the developing gyrencephalic brain

doi: 10.1186/s40478-026-02257-3

Figure Lengend Snippet: Postnatal GABAergic maturation occurs in human hippocampus. Schematic of study design, sample collection protocol, and sample demographics ( A ). Quantitative PCR analysis for transcripts Slc12a2 , Slc12a5 , and Stk39 ( B ) across postconceptional age (PCA) presented as fold change from youngest sample. Ratio of Slc12a5 to Slc12a2 as a function of PCA ( C ). Representative western blots of hippocampal lysates ( D ), quantified in ( E ) as a function of PCA. SPAK, NKCC1, and KCC2 all demonstrate positive linear correlations with PCA. In all graphs, linear regression analysis was used. R 2 values are shown on the graphs along with a p-value for whether the slope is non-zero

Article Snippet: NKCC1 , Rabbit , Synthetic Arg77 of mouse NKCC1 , Cell Signaling Technology , 14,581 , KO lysate , 1:1000 , .

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Temporal and cell-type specific SPAK-NKCC1 disruption following severe TBI in the developing gyrencephalic brain

doi: 10.1186/s40478-026-02257-3

Figure Lengend Snippet: Proxy markers of transition to inhibitory GABA increase perinatally in swine, particularly across cortex. A , schematic of samples represented across gestation and postnatal ages. B , regions collected for analysis. C , quantitative PCR analysis of samples across ages (ED36, ED90, ED105, Infant, Toddler) and regions (cortex, hippocampus, thalamus) for Slc12a2 (encoding NKCC1), Slc12a5 (encoding KCC2), and Stk39 (encoding SPAK). 2-way ANOVA for Stk39 expression across ages and regions: main effect of age p = 0.0007, main effect of region p = 0.0008; post-hoc multiple t-test p < 0.01. D, representative western blots for protein and phospho protein abundance, total protein stain as loading control. E, quantitation of western lots for samples across ages and regions for total protein (left) and ratio of phosphorylated to total protein (right). KCC2: 2-way ANOVA main effect of age p = 0.0002, main effect of region p = 0.0043; post-hoc multiple t-test ** p < 0.01. SPAK: 2-way ANOVA main effect of age p < 0.0001, main effect of region p-0.008; post-hoc multiple t-test * p < 0.05,**** p < 0.0001. pKCC2/KCC2: 2-way ANOVA main effect of age p = 0.0015, main effect of region n.s.; post-hoc multiple t-test ** p < 0.01. pSPAK/SPAK: 2-way ANOVA main effect of age p < 0.0001, main effect of region p = 0.0104; post-hoc multiple t-test ** p < 0.01. pNKCC1/NKCC1: 2-way ANOVA main effect of age p = 0.0012, main effect of region p = 0.018; post-hoc multiple t-test p = 0.0649

Article Snippet: NKCC1 , Rabbit , Synthetic Arg77 of mouse NKCC1 , Cell Signaling Technology , 14,581 , KO lysate , 1:1000 , .

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Quantitative Proteomics, Staining, Control, Quantitation Assay

Journal: Acta Neuropathologica Communications

Article Title: Temporal and cell-type specific SPAK-NKCC1 disruption following severe TBI in the developing gyrencephalic brain

doi: 10.1186/s40478-026-02257-3

Figure Lengend Snippet: Severe TBI induces a reversion of markers of immature GABA in “infants” and upregulation of maturation in “toddlers”. Representative patterns of hypoxic-ischemic injury at the level of the somatosensory cortex between ages ( A , yellow = injured tissue). Quantitative PCR analysis for transcripts Slc12a2 ( B ), Slc12a5 ( C ), and Stk39 ( D ) due to age and TBI injuries in piglets, presented as fold change from sham infant piglets. Two-way ANOVA results are below graph, and post-hoc multiple t-tests are included on graphs where applicable (* p < 0.05, ** p < 0.01). E , Study design and ROI selection for GeoMx Spatial Transcriptomics. F , selected relative expression of Slc12a2 and Slc12a5 across regions from sham animals and animals with severely injured tissue. Three-Way ANOVA performed to analyze transcriptional levels across gene, region, and injury. Western blots from cortical lysates in infant (G) and toddler (H) piglets probed for pNKCC1, NKCC1, TrkB, SPAK, pKCC2, and KCC2, normalized to beta actin, quantified by band densiometry and graphed with regard to sham v. TBI (I), time for TBI injuries (J), and hemisphere for TBI injuries (K). Two-way ANOVA results are below graph, and post-hoc multiple t-tests are included on graphs where applicable with exact p values

Article Snippet: NKCC1 , Rabbit , Synthetic Arg77 of mouse NKCC1 , Cell Signaling Technology , 14,581 , KO lysate , 1:1000 , .

Techniques: Real-time Polymerase Chain Reaction, Selection, Spatial Transcriptomics, Expressing, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Temporal and cell-type specific SPAK-NKCC1 disruption following severe TBI in the developing gyrencephalic brain

doi: 10.1186/s40478-026-02257-3

Figure Lengend Snippet: Protein expression of NKCC1, KCC2, and SPAK in the hippocampus and thalamus were minimally sensitive to severe TBI injuries in piglets despite persistent transcriptional changes. Representative patterns of minimal hypoxic-ischemic injury at the levels of the hippocampus and thalamus between ages ( A , yellow = injured tissue). Quantitative PCR analysis for transcripts Slc12a2 ( B , E ), Slc12a5 ( C , F ) , and Stk39 ( D , G ) due to age and TBI injuries in piglets, presented as fold change from sham infant piglets in hippocampus ( B–D ) and thalamus ( E–G ). Two-way ANOVA results are below graph, and post-hoc multiple t-tests are included on graphs where applicable (* p < 0.05, ** p < 0.01). Western blots for all biological replicates for infant and toddler piglets receiving sham or severe TBI injuries in hippocampus and thalamus ( H ) and quantitated below ( I , J )

Article Snippet: NKCC1 , Rabbit , Synthetic Arg77 of mouse NKCC1 , Cell Signaling Technology , 14,581 , KO lysate , 1:1000 , .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: Mycobacterium tuberculosis modulates phosphorylation of host ATP6V1E1 to promote intracellular survival

doi: 10.1038/s41467-026-69331-1

Figure Lengend Snippet: a Quantification of western blots to screen Kinase library regulated ATP6V1E1 phosphorylation. b In vitro phosphorylation assay of ATP6V1E1. The proteins of GFP-BMX, Flag-ATP6V1E1 and HA-ATP6V1E1 Y56/Y57A were overexpressed and purified in HEK293T cells. c–e Detection of ATP6V1E1 phosphorylation in macrophages transfected with Bmx siRNA ( c ), treated with BMX inhibitor (10 nM) ( d ) or in Bmx +/- macrophages ( e ), then infected with H37Rv (MOI = 5) for indicated times. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. f Subcellular fractionation of macrophages from WT or Bmx +/- mice or WT macrophages under the treatment with BMX-IN (10 nM) for 1 h. Cytosolic (C) and membrane (M) fractions were immunoblotted with antibodies against the V1 domain subunit ATP6V1E1 and the V0 domain subunit ATP6V0d1. α-Tubulin and V0d1 serve as loading controls for cytosolic and membrane fractions, respectively. g Representative LysoTracker staining of macrophages transfected with scrambled or Bmx -specific siRNA and then infected with H37Rv (MOI = 5) for the indicated times. Scale bar, 10 μm. h Quantification of LysoTracker fluorescence intensity (mean ± SEM). i Intracellular CFU in macrophages transfected scrambled or Bmx -specific siRNA and then infected with H37Rv (MOI = 5) for the indicated times (mean ± SEM). j Intracellular CFU in macrophages from WT and Atp6v1e1 +/- mice treated with BMX inhibitor (10 nM) and infected with H37Rv (MOI = 5) for the indicated times (mean ± SEM). k–l Intracellular CFU and bacterial survival in macrophages from WT or Bmx +/- mice infected with H37Rv or H37RvΔChp2 for the indicated times (MOI = 5) (mean ± SEM). ( m )H&E staining of lung tissues from WT and Bmx +/- mice infected with H37Rv by intranasal infection ( ~ 200 CFU) for 4 weeks. (mean ± SEM, N = 3 mice) #1-3 indicate three representative lung tissues. Scale bar, 5000 μm. n Quantification of the histopathology score in (m) (mean ± SEM). o Bacterial CFUs in the lungs of WT and Bmx +/- mice in (m) (mean ± SEM). p Acid-fast staining of the lung tissues of WT and Bmx +/- mice in (m). Scale bar, 100 μm. Data in ( a–m ) are representative of one experiment with at least three independent biological replicates ( h–l , n , o , n = 3). Two-tailed unpaired Student’s t test (h-l and n) was used for statistical analysis. Two-sided Mann-Whitney U test ( o ) was used for statistical analysis.

Article Snippet: Anti-V0d1 antibody(18274-1-AP), anti-tubulin antibody (80762-1-RR), anti- Na-K antibody (13884-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Phospho-proteomics, In Vitro, Purification, Transfection, Infection, Fractionation, Membrane, Staining, Fluorescence, Histopathology, Two Tailed Test, MANN-WHITNEY