Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: anti-pSTAT6 149 Sm-conjugated Clone 18 , Fluidigm , Cat No.3149004A.

Techniques: Recombinant, Virus, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Extraction, Mutagenesis, Isolation, Reverse Transcription, Luminex, Transfection, Amplification, Software, Variant Assay

Journal: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-human phospho-STAT6 (Tyr641) (clone 18) , Fluidigm , Cat#3168012A; RRID:AB_2811103.

Techniques: Recombinant, Mass Cytometry, Software

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet: KLF4 is associated with sleep deprivation-induced ADRB2 modulation of macrophage prompting on proliferation, migration, and invasion of LLC-LUC cells (A) Differentially expressed Top20 genes in metastatic lung tissues of NC group and sleep deprivation group. (B) The expression of top 5 upregulated genes in tumor tissues was analyzed by qRT-PCR. (C) KLF4 expression was detected by ADRB2-specific agonist ISO and ADRB2 antagonist ICI118,551 in macrophages treated with IL-4 in vitro . (D) The effect of ADRB2 agonist on CD206 expression in IL-4-induced KLF4-knockdown macrophages. (E) EdU assays showed the proliferation of tumor cells in the coculture system with 30% CM. CM derived from IL-4 and IL-4 + ISO induced supernatant of macrophages with KLF4 low and high expression. (F) The migration ability of tumor cells was determined by wound healing assays in the indicated coculture medium. (G) Transwell assays demonstrated invasion of tumor cells in the indicated coculture system. (H) Representative IHC images of p-JAK1 and p-STAT6 in xenograft tumors. (I) The phosphorylation of JAK1 and STAT6 expression in RAW264.7 treated with ADRB2 agonist or antagonist via Western blotting. (J) Immunoblotting of KLF4 in RAW264.7 treated with different conditions. Data represented as mean ± SD. Statistical analysis was performed using Student’s t test or Mann-Whitney U test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The paraffin sections of mice tumor tissues were subjected to baking, dewaxing, dehydration, elimination of endogenous peroxidase activity, tissue pressure-resistant antigen repair, 5% BSA blocking, dropping of rabbit polyclonal antibody against KLF4 (Proteintech), rabbit polyclonal antibody against CD31 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay), rabbit polyclonal antibody against p-STAT6 (ImmunoWay), PBS washing at 4°C overnight, incubation with secondary antibody at 37°C for 35 min, PBS washing, and staining.

Techniques: Migration, Expressing, Quantitative RT-PCR, In Vitro, Knockdown, Derivative Assay, Phospho-proteomics, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet: NE level is elevated in NSCLC patients with sleep deprivation and is associated with a poor prognosis (A) The concentration of NE in serum of the NSCLC patients, determined by ELISA assay. (B) Representative IHC images and quantitative analysis of ADRB2 in the NSCLC patients. (C) Representative IHC images and quantitative analysis of KLF4 in the NSCLC patients. (D) Investigation into the association between ADRB2 expression and immune cell infiltration in patients diagnosed with LUAD. (E) Analysis of the correlation between KLF4 and immune cell infiltration in LUAD patients. (F–H) Correlation analysis between ADRB2, KLF4, JAK1, and STAT6 expression. (I and J) Correlation analysis between KLF4, JAK1, and STAT6 expression. (K and L) Correlation analysis between JAK1, STAT6, and pro-tumoral macrophages. Data represented as mean ± SD. Statistical analysis was evaluated using Student’s t test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The paraffin sections of mice tumor tissues were subjected to baking, dewaxing, dehydration, elimination of endogenous peroxidase activity, tissue pressure-resistant antigen repair, 5% BSA blocking, dropping of rabbit polyclonal antibody against KLF4 (Proteintech), rabbit polyclonal antibody against CD31 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay), rabbit polyclonal antibody against p-STAT6 (ImmunoWay), PBS washing at 4°C overnight, incubation with secondary antibody at 37°C for 35 min, PBS washing, and staining.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet:

Article Snippet: The paraffin sections of mice tumor tissues were subjected to baking, dewaxing, dehydration, elimination of endogenous peroxidase activity, tissue pressure-resistant antigen repair, 5% BSA blocking, dropping of rabbit polyclonal antibody against KLF4 (Proteintech), rabbit polyclonal antibody against CD31 (Proteintech), rabbit polyclonal antibody against p-JAK1 (ImmunoWay), rabbit polyclonal antibody against p-STAT6 (ImmunoWay), PBS washing at 4°C overnight, incubation with secondary antibody at 37°C for 35 min, PBS washing, and staining.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Phagocytosis Assay, Sequencing, Software

Journal: Cell Death and Differentiation

Article Title: PP2Acα promotes macrophage accumulation and activation to exacerbate tubular cell death and kidney fibrosis through activating Rap1 and TNFα production

doi: 10.1038/s41418-021-00780-5

Figure Lengend Snippet: a , b Western blot assay ( a ) and semi-quantitative analysis ( b ) showing that deletion of PP2Acα enhanced LPS-stimulated Stat6 phosphorylation in BMDMs. * p < 0.05, n = 3. Data are presented as means ± SEM. c , d Western blot assay ( c ) and quantitative analysis ( d ) showing that overexpression of PP2Acα could inhibit Stat6 phosphorylation caused by PP2Acα ablation. * p < 0.05, n = 3. # p < 0.05, n = 3. Data are presented as means ± SEM. e Real-time qRT-PCR analysis showing the mRNA abundance of Tnfα in BMDMs. * p < 0.05, n = 6. # p < 0.05, n = 6. Data are presented as means ± SEM. f ELISA assay showing the protein abundance of TNFα in the cultural media of AS1517499-treated BMDMs. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. g , h Representative PI staining images ( g ) and quantitative analysis ( h ) showing that the conditioned media of AS1517499-treated macrophages caused more tubular cell death. Scale bar, 100 μm. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. i Real-time qRT-PCR analysis showing the mRNA abundance of Stat6 in scramble siRNA and Stat6 siRNA-transfected BMDMs. * p < 0.05, n = 3. Data are presented as means ± SEM. j ELISA analysis showing the protein abundance of TNFα in the cultural media of Stat6 siRNA-transfected BMDMs. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. k , l Representative PI staining images ( k ) and quantitative analysis ( l ) showing that the conditioned media of Stat6 siRNA-transfected macrophages caused more tubular cell death. Scale bar, 100 μm. * p < 0.05, n = 3. # p < 0.05, n = 3. $ p < 0.05, n = 3. Data are presented as means ± SEM. m Representative immune staining images showing the induction of p-Stat6 (T645) in F4/80-positive macrophages within MФ-PP2Acα −/− fibrotic kidneys. White arrows indicate double-staining-positive cells. Scale bar, 10 μm. n , o Quantitative analysis for p-Stat6 (T645) and F4/80 double-positive cells in IRI ( n ) and UUO ( o ) kidney tissues. * p < 0.05, n = 3. Data are presented as means ± SEM. p , q Western blot analyses ( p ) and quantitative determination ( q ) showing that deletion of PP2Acα could induce the phosphorylation of Stat6 in macrophages sorted from MФ-PP2Acα −/− IRI kidneys. * p < 0.05, n = 3. Data are presented as means ± SEM. r , s Western blot analyses ( r ) and quantitative determination ( s ) showing that deletion of PP2Acα could induce the phosphorylation of Stat6 in macrophages sorted from MФ-PP2Acα −/− UUO kidneys. * p < 0.05, n = 3. Data are presented as means ± SEM.

Article Snippet: The primary antibodies were anti-PP2Ac (cat: 2038, Cell Signaling Technology, Boston, MA, USA, 1:1000), anti-PP2Acα (cat: ab106262, Abcam, Cambridge, UK, 1:1000), anti-PP2Acβ (cat: ab168371, Abcam, 1:1000), anti-methyl-PP2Ac (L309) (cat: ab66597, Abcam, 1:1000), anti-Itgb2 (cat: ab119830, Abcam, 1:1000), anti-Epac1 (cat: ab124162, Abcam, 1:1000), anti-FN (cat: F3648, Sigma-Aldrich, 1:10000), anti-Stat6 (cat: ab32520, Abcam, 1:1000), anti-p-Stat6 (T645) (cat: BS4186, Bioworld Technology, Nanjing, China, 1:1000), anti-Rap1a/b (cat: 4938, Cell Signaling Technology, 1:1000), anti-tubulin (cat: sc53646, Santa Cruz Biotechnology, 1:10000), and anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, 1:5000).

Techniques: Western Blot, Over Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Transfection, Double Staining