c29h4 cell signaling technology  (Cell Signaling Technology Inc)

Journal: The FASEB Journal

Article Title: KCNB1‐Leptin receptor complexes couple electric and endocrine function in the melanocortin neurons of the hypothalamus

doi: 10.1096/fj.202401931r

Figure Lengend Snippet: FIGURE 6 Canonical and non-canonical leptin pathways are altered in NULL. (A) Graphical illustration of LepR signaling. Canonical (JAK2/STAT3) and non-canonical PI3K/Akt/FOXO1 and ERK pathways are indicated. (B) Representative Western blot and densitometric quantification of the long isoform leptin receptor (LepR) in hypothalamic lysates of WT and NULL mice. (C) Representative Western blot of STAT3 phosphorylated at Y705 (pSTAT3) and total STAT3 protein (STAT3) in hypothalamic lysates of WT and NULL mice in absence/presence of leptin. (D) Densitometric quantifications of the fraction of pSTAT3/STAP calculated from Westerns as that in (C). (E) Representative Western blot of phosphorylated FOXO1 at S256 (pFOXO1) and total FOXO1 protein (FOXO1) in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin. (F) Densitometric quantifications of pFOXO1/FOXO1 calculated from Westerns as those in (E). (G) ELISA quantifications of phosphorylated Akt at S473 (pAkt) over total Akt protein (pAkt/Akt) in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin. (H) Representative Western blots of phosphorylated ERK1/2 at T202/T204 (pERK) and total ERK1/2 (ERK) protein in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin and corresponding densitometric quantifications of the fraction of phosphorylated ERK1/2 over total ERK1/2 (pERK/ERK). (I) ELISA quantifications of αMSH in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin. Western blot loading controls were actin and Bradford colorimetric assay. Animals were injected with either vehicle or 1.0 mg/kg body weight leptin and sacrificed 90 min later. *p < .05 (ANOVA with Tukey's post hoc).

Article Snippet: Reagent or resource Source Identifier Antibodies POMC (D3R1U) Rabbit mAb Cell Signaling Technology #23499 c- Fos (9F6) Rabbit mAb Cell Signaling Technology #2250 Stat3 (124H6) Mouse mAb Cell Signaling Technology #9139 Phospho- Stat3 (Tyr705) (M9C6) Mouse mAb Cell Signaling Technology #4113 FoxO1 (C29H4) Rabbit mAb Cell Signaling Technology #2880 Phospho- FoxO1 (Ser256) (E1F7T) Rabbit mAb Cell Signaling Technology #84192 p44/42 MAP kinase (phosphorylated Erk1/2) Cell Signaling Technology Cat# 9101 p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb Cell Signaling Technology Cat# 4695 Integrin beta- 5 (D24A5) Rabbit mAb Cell Signaling Technology Cat# 3629 Integrin beta- 1 antibody Cell Signaling Technology Cat #4706 Alpha MSH polyclonal rabbit Bioss Antibodies bs- 1848R AgRP polyclonal antibody Thermo Fisher PA5- 118934 Leptin receptor polyclonal antibody Thermo Fisher PA1- 053 Leptin receptor recombinant Rabbit monoclonal antibody Thermo Fisher MA5- 32685 (NIH), Grant/Award Number: R01AG060919; National Science Foundation (NSF), Grant/Award Number: 2030348 upon leptin stimulation.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Injection

Journal: Nature Communications

Article Title: Quinolinate promotes macrophage-induced immune tolerance in glioblastoma through the NMDAR/PPARγ signaling axis

doi: 10.1038/s41467-023-37170-z

Figure Lengend Snippet: a Macrophages obtained from C57BL/6 mice were polarized towards the M2 phenotype ±QA (20 µM) or the PPARγ agonist troglitazone (Trog; 5 µM) and evaluated for the indicated proteins by western blot, which is representative of three biologically independent experiments. b Macrophages obtained from C57BL/6 mice were polarized towards the M2 phenotype ±Trog ( n = 5), GW9662 (PPARγ antagonist; n = 3) and/or QA (20 µM; n = 5) and analyzed for M2 macrophage markers (CD45 + CD11b + F4/80 + CD206 + ) by flow cytometry. All samples were biologically independent. Numbers represent mean ± SD. c PPARγ transcriptional activity was evaluated in the murine macrophage cell line IC-21 polarized towards the M2 phenotype ±siRNA of indicated proteins by PPARγ transcriptional activity ELISA ( n = 5 biologically independent samples). d C57BL/6 mouse-derived macrophages were polarized towards the M2 phenotype ±QA (20 µM) and evaluated for the indicated proteins by western blot, which is representative of 3 biologically independent experiments. e Macrophages were cultured in the presence or absence of QA ± the Foxo1 inhibitor AS-1842856 (0.1 µM) and evaluated for M2 macrophage markers (CD45 + CD11b + F4/80 + CD206 + ) by flow cytometry. Numbers represent mean ± SD ( n = 4 biologically independent samples/group). f Chromatin immunoprecipitation (ChIP) was performed on C57BL/6 mouse-derived macrophages polarized towards the M2 phenotype and evaluated alone (gray), with QA (red) or with Trog (blue). ChIP was performed using antibodies against PPARγ, Foxo1, and histone 3 (positive control), and IgG antibody (negative control). After IP and DNA purification, the PPARγ exon (202 bp in length) was analyzed using qPCR and gel electrophoresis. Box plots represent interquartile range, line between the data points represents mean, and whiskers represents SE ( n = 4 biologically independent samples/group). Statistics: One-way ANOVA followed by Tukey’s multiple comparisons test ( b , c ), two-tailed Student’s t -test ( e , f ). All tests were performed at 95% confidence interval. Source data are provided as a source data file.

Article Snippet: Chromatin was sonicated and then incubated with a polyclonal antibody against PPARγ (81B8 and C26H12) or FoxO1 (C29H4) Cell Signaling Technology (Danvers, MA) for immunoprecipitation.

Techniques: Western Blot, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Chromatin Immunoprecipitation, Positive Control, Negative Control, DNA Purification, Nucleic Acid Electrophoresis, Two Tailed Test

Journal: Nature Communications

Article Title: Quinolinate promotes macrophage-induced immune tolerance in glioblastoma through the NMDAR/PPARγ signaling axis

doi: 10.1038/s41467-023-37170-z

Figure Lengend Snippet: a M2 macrophages were polarized ±QA, gated for macrophage markers (CD45 + CD11b + F4/80 + ) and CD206 + ve with (green) or without (red) QA or CD206-ve with (orange) or without (dark green) QA, and evaluated for expression of the NMDA receptor 1 (NMDAR1; n = 2 biologically independent samples/group). b M2 macrophages were cultured in ±QA, L-AP5 (NMDAR1 inhibitor), or NMDA and cells were gated for M2 macrophages markers (CD45 + CD11b + F4/80 + CD206 + ). Numbers represent mean ± SD ( n = 3 biologically independent samples/group). c M2 macrophages were cultured in ±QA, NMDA, or Trog, and indicated proteins were evaluated by western blot, which is representative of three biologically independent experiments. d Schematic depicting the proposed mechanism of QA-induced M2 macrophage polarization. QA binds to the NMDA receptor of macrophages (1), leading to phosphorylation of Foxo1 (2). Phosphorylated Foxo1 is retained in the cytoplasm and destined for ubiquitination. Loss of nuclear Foxo1, a negative regulator of the PPARγ promoter, leads to increased PPARγ expression and transcriptional programs designed to promote macrophage polarization towards the M2 phenotype (3). Statistics: Two-tailed Student’s t test at 95% confidence interval ( b ). Source data are provided as a source data file.

Article Snippet: Chromatin was sonicated and then incubated with a polyclonal antibody against PPARγ (81B8 and C26H12) or FoxO1 (C29H4) Cell Signaling Technology (Danvers, MA) for immunoprecipitation.

Techniques: Expressing, Cell Culture, Western Blot, Phospho-proteomics, Ubiquitin Proteomics, Two Tailed Test