Journal: ImmunoHorizons

Article Title: The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4 + Th2 Differentiation

doi: 10.4049/immunohorizons.1700017

Figure Lengend Snippet: ( A ) Flow cytometry of kinetic changes in CD25 protein expression of activated YFP + , YFP − , and WT CD4 + T cells on days 0–5. ( B ) p-Stat5, p-Stat6, total Stat5, and total Stat6 protein levels in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. ( C ) Blimp1 protein expression in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data are representative of two (B) or three (A and C) independent experiments. nonpolarizing conditions on days 0 to 5. ( E ) Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2 . Data are combined from three (B, C, and E) or four [(A), right panel and (D)] independent experiments, along with representative flow cytometry plots (A, left panel). Error bars represent mean + SEM of three (B, C, and E) or four [(A), right panel] independent experiments. The p values in (D) were calculated based on YFP + versus YFP − and WT. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with the Tukey multiple-comparisons test.

Article Snippet: The membranes were blocked with 5% BSA in TBST and probed with anti-STAT5 (1:250), anti–p-STAT5 pY694 (1:500), anti-STAT6 (1:500), anti–p-STAT6 pY641 (1:250) (BD Biosciences), anti–β-actin (1 μg/ml) (Sigma-Aldrich) and anti-HuR clone 3A2 (1 μg/ml).

Techniques: Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: ImmunoHorizons

Article Title: The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4 + Th2 Differentiation

doi: 10.4049/immunohorizons.1700017

Figure Lengend Snippet: ( A ) YFP + , YFP − , or WT CD4 + T cells were stimulated or not with 100 U/ml rIL-4. Five days postactivation, cells were harvested and restimulated with PMA and ionomycin for 5 h, and cytokine production was assessed by intracellular cytokine staining. ( B ) p-Stat5 and p-Stat6 levels in YFP + , YFP − , and WT CD4 + T cells activated in the presence of IL-4. ( C ) CD25 expression in YFP + , YFP − , and WT CD4 + T cells activated in the presence or absence of 1000 U/ml rIL-4 for 5 d. Data (mean + SEM) are a representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with the Tukey multiple-comparisons test.

Article Snippet: The membranes were blocked with 5% BSA in TBST and probed with anti-STAT5 (1:250), anti–p-STAT5 pY694 (1:500), anti-STAT6 (1:500), anti–p-STAT6 pY641 (1:250) (BD Biosciences), anti–β-actin (1 μg/ml) (Sigma-Aldrich) and anti-HuR clone 3A2 (1 μg/ml).

Techniques: Staining, Expressing

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