ire proteintech 27528 1 ap  (Proteintech)

Journal: Nature Communications

Article Title: Stress induces oxytocin-Gαi-dependent remodeling of astrocytes to shape neuronal response in the amygdala

doi: 10.1038/s41467-025-68114-4

Figure Lengend Snippet: a Imaging of CeL/C astrocytes using targeted expression of eGFP using rAAV-GFAP-eGFP. b Representative micrograph showing synapses and surrounding astrocytic processes following IMARIS-based 3D reconstruction. Arrowheads show a putative synapse contacted by an astrocyte. eGFP: astrocyte, vGlut1/2: post-synaptic, Homer1: pre-synaptic c Quantification of the contacts between synapses and astrocyte processes per reconstructed astrocyte. n = 5 animals in each group. d Analysis of BLA-to-CeL/C synaptic transmission using optogenetic stimulation of the BLA axons and recordings of the EPSCs in CeL/C neurons in naïve and stressed mice. Injection of AAV-CaMKII-ChR2 was performed in the BLA of WT mice, and a second pair of injection containing rAAV-GFAP-Cre was done in the CeL/C of OTR-lox mice. 3 weeks after, we measured the currents evoked by BLA neurons activation in CeL/C neurons of presynaptic BLA neurons. Lower right panel: representative recording of EPSCs in CeL/C neurons evoked during photostimulation. e , f Time course and quantification of photostimulation-evoked EPSCs amplitude. n (neurons, mice): WT naive=14, 5, WT stress=19, 5, GFAP OTR cKO naïve=17, 9, GFAP OTR cKO stress=13, 5. g Experimental approach to record K + currents in CeL/C astrocytes following optogenetic stimulation of BLA neurons in naïve and stressed mice. AAV-CaMKII-ChR2 was injected in the BLA of mice, and 3 weeks after, the currents evoked by BLA neuron activation were measured in CeL/C astrocytes. Lower right panel: representative recording of photstimulation-evoked currents in control condition, after the application of Ba 2+ , and the subtraction of both (I K ). h , i Time course and quantification of photostimulation-evoked astrocytic current induced by BLA neurons stimulation before (black) and after a 15-min incubation in Ba 2+ (200µM, dark grey). The subtracted K + current is shown in light grey. n (astrocyte, mice): WT naive=13,6, WT stress=11, 8, GFAP OTR cKO naïve=11, 6, GFAP OTR cKO stress=8, 6. j Quantification of K ir 4.1 levels in naïve and stressed mice using 1mm punches of mice CeA was done on 400µm slices from naïve or stressed mice. k Image of representative Western blot reacted with antibody against K ir 4.1 in six animals from the indicated experimental groups. l Quantification of K ir 4.1 levels in amygdala punches. n naïve =6, n stress =8 mice. Data are expressed as mean across animals ± SEM for ( c ) and ( l ), and as mean across cells ± SEM for ( e ), ( f ), ( h ), and ( i ). Detailed statistics can be found in Source Data Table . * p < 0.05, ** p < 0.01. Drawings created in BioRender: Charlet, A. (2025) https://BioRender.com/bkv8qa0 .

Article Snippet: After 3x10min rinses in 9 mM NaCl Tris-HCl Buffer + 0.1% Tween 20 (TBST), membranes were blocked in TBST + 5% milk for 1 h at room temperature (RT) and incubated overnight at 4 °C sequentially with the following primary antibodies: anti-K ir 4.1 (1:1000, Rabbit; Alomone, #APC-035), anti-actin or anti-Tom20 in TBST + 5% milk.

Techniques: Imaging, Expressing, Transmission Assay, Injection, Activation Assay, Control, Incubation, Western Blot

Journal: Nature Communications

Article Title: 6-Phosphogluconate dehydrogenase promotes mitochondrial fusion and immune suppression in tumor-associated monocytic suppressor cells

doi: 10.1038/s41467-025-68102-8

Figure Lengend Snippet: A MDSCs were generated in vitro using IL-6 (40 ng/mL) and GM-CSF (40 ng/mL) from bone marrow (BM) of Pgd fl/fl and Pgd fl/fl LysM Cre mice or wild type (WT) mice with 6AN (5 µM) or vehicle. Sorted MDSCs on day 4 were analyzed for 1318 signaling target proteins by Phospho-Explorer Antibody Array (Full moon BioSystem). B Oxidative PPP checkpoints were blocked by shRNA in WT BM-derived MDSCs. Phospho-IRS-1 S307 levels were examined on sorted M-MDSCs. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. C 6PG (10, 100, 1000 µM) was added to MDSC cell free lysates for 30 min and IRS-1 S307 phosphorylation measured by western blot. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. D , E Immunoprecipitated JNK1 and IRS-1 (substrate) were incubated with increasing 6PG concentrations (10, 100, 1000 µM) in an in vitro kinase assay ( D ). In the same experiment, JNK1-IRS-1 binding was evaluated by co-immunoprecipitation (Co-IP) using anti-JNK1 ( E ). Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. F Serine to Alanine (S307A) site-specific mutation in IRS-1 was conducted in M-MDSCs (generated as in ( A )). Flowcytometry-sorted M-MDSCs were co-cultured with anti-CD3/anti-CD28 activated T cells (CFSE-labeled) at a 1:8 (M-MDSC: T cell) ratio. T cell proliferation was evaluated at 72 h. n = 3; One-way ANOVA. G , H M-MDSCs were generated as in ( A , F ). Mitochondrial ROS (mROS) production ( G ) and Arginase 1 expression ( H ) was examined on M-MDSC by flow cytometry. n = 3; One-way ANOVA. I Sorted M-MDSCs were prepared as in ( A , F ). Levels of PI3K (top), AKT (middle) and Drp1 S616 (bottom) phosphorylation were determined by western blot analysis. Blots were processed parallel to samples from the same experiment. Result is representative of two repeats. J – L MDSCs were generated as in ( A , F ), with N-Acetylcysteine (NAC: 0.5 mM) or vehicle (PBS). mROS production in M-MDSCs ( J ), suppressive capacity in co-culture with T cells ( K ) and Arginase expression levels ( L ) determined. n = 3; One-way ANOVA. All data are shown as mean ± SEM.

Article Snippet: The IRS-1 sequence containing a N-terminal HA tag was obtained by PCR using mouse IRS-1 cDNA as a template and cloned into the appropriate multiple cloning sites of the expression vector pcDNA 3.1(+) (CUSABIO).

Techniques: Generated, In Vitro, Ab Array, shRNA, Derivative Assay, Phospho-proteomics, Western Blot, Immunoprecipitation, Incubation, Kinase Assay, Binding Assay, Co-Immunoprecipitation Assay, Mutagenesis, Cell Culture, Labeling, Expressing, Flow Cytometry, Co-Culture Assay

Journal: Nature Communications

Article Title: 6-Phosphogluconate dehydrogenase promotes mitochondrial fusion and immune suppression in tumor-associated monocytic suppressor cells

doi: 10.1038/s41467-025-68102-8

Figure Lengend Snippet: A MDSCs were generated in vitro using IL-6 (40 ng/mL) and GM-CSF (40 ng/mL) from bone marrow (BM) of Pgd fl/fl and Pgd fl/fl LysM Cre mice or wild type (WT) mice with 6AN (5 µM) or vehicle. Sorted MDSCs on day 4 were analyzed for 1318 signaling target proteins by Phospho-Explorer Antibody Array (Full moon BioSystem). B Oxidative PPP checkpoints were blocked by shRNA in WT BM-derived MDSCs. Phospho-IRS-1 S307 levels were examined on sorted M-MDSCs. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. C 6PG (10, 100, 1000 µM) was added to MDSC cell free lysates for 30 min and IRS-1 S307 phosphorylation measured by western blot. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. D , E Immunoprecipitated JNK1 and IRS-1 (substrate) were incubated with increasing 6PG concentrations (10, 100, 1000 µM) in an in vitro kinase assay ( D ). In the same experiment, JNK1-IRS-1 binding was evaluated by co-immunoprecipitation (Co-IP) using anti-JNK1 ( E ). Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. F Serine to Alanine (S307A) site-specific mutation in IRS-1 was conducted in M-MDSCs (generated as in ( A )). Flowcytometry-sorted M-MDSCs were co-cultured with anti-CD3/anti-CD28 activated T cells (CFSE-labeled) at a 1:8 (M-MDSC: T cell) ratio. T cell proliferation was evaluated at 72 h. n = 3; One-way ANOVA. G , H M-MDSCs were generated as in ( A , F ). Mitochondrial ROS (mROS) production ( G ) and Arginase 1 expression ( H ) was examined on M-MDSC by flow cytometry. n = 3; One-way ANOVA. I Sorted M-MDSCs were prepared as in ( A , F ). Levels of PI3K (top), AKT (middle) and Drp1 S616 (bottom) phosphorylation were determined by western blot analysis. Blots were processed parallel to samples from the same experiment. Result is representative of two repeats. J – L MDSCs were generated as in ( A , F ), with N-Acetylcysteine (NAC: 0.5 mM) or vehicle (PBS). mROS production in M-MDSCs ( J ), suppressive capacity in co-culture with T cells ( K ) and Arginase expression levels ( L ) determined. n = 3; One-way ANOVA. All data are shown as mean ± SEM.

Article Snippet: The IRS-1 sequence containing a N-terminal HA tag was obtained by PCR using mouse IRS-1 cDNA as a template and cloned into the appropriate multiple cloning sites of the expression vector pcDNA 3.1(+) (CUSABIO).

Techniques: Generated, In Vitro, Ab Array, shRNA, Derivative Assay, Phospho-proteomics, Western Blot, Immunoprecipitation, Incubation, Kinase Assay, Binding Assay, Co-Immunoprecipitation Assay, Mutagenesis, Cell Culture, Labeling, Expressing, Flow Cytometry, Co-Culture Assay