rabbit anti human phosphorylated stat1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.64898/2025.12.08.692940

Figure Lengend Snippet: PI3K/AKT-mTOR but not STAT1 signaling pathways downstream of IFN-I are implicated in the synergy between IFN-I and C. trachomatis . (A) HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h followed by the detection of activation and expression of STAT1 via immunoblot. The results are representatives of three independent experiments. (B) siRNA against STAT1 or irrelevant oligonucleotides were transfected into HeLa cells for 24 h prior to incubation with IFNβ alone (left graph) or with IFNβ and C. trachomatis (right graph). The transcriptional level of STAT1 (left graph) was measured by real-time RT-qPCR and normalized to actin transcript following the 2 -ΔΔCt method. The data are presented as relative mRNA levels compared to untreated cells and shown as the mean±SE with individual values of three experiments. P-value of Student’s unpaired t-test is shown (* for p < 0.05). Intracellular IL6 protein (right panel) was determined by flow cytometry using anti-human IL6-PC7 antibody. The results of four independent experiments are shown. C-D) HeLa cells were pre-incubated with wortmannin (5 μM) (C) or SB203580 (10 μM) (D) for 1 h. Cells were then treated with IFNβ and/or C. trachomatis for 24 h in the presence of these inhibitors prior to brefeldin A addition for 6 h. Intracellular IL6 expression was analyzed by flow cytometry using anti-human IL6-PC7 antibody. The histograms are the representatives of two (C) or four experiments (D), respectively. The results of the four independent experiments are shown as a violin plot in (D), with p-value of a Student’s unpaired t-test (* for p< 0.05). (E) Same as in D using mTOR inhibitors, rapamycin and torin1 (1 μM). The results of five independent experiments and p-value of a Student’s unpaired t-test are shown (* for p< 0.05). (F) HeLa cells were treated with pharmacological inhibitors (upper panels) or transfected with siRNA (lower panel) as described above, followed by IFNβ and/or C. trachomatis treatment for 24 h. TLR3 transcripts was measured by rea-time RT-qPCR as above. Data from three independent experiments and p-values of Student’s unpaired t-test are shown (* p< 0.05, ** p<0.01, *** p<0.001 and **** p<0.0001). (G) The cells were incubated with SB203580 (10 μM) for 1 h before addition of IFNβ for 30 min. Phosphorylation of mTOR, AKT and p38 was detected by immunoblot. The results are representatives of three independent experiments.

Article Snippet: Primary antibodies used were rabbit anti-human STAT1 (#9172), Erk (#4695), p38 (#9212), AKT (#9272), rabbit anti-human phosphorylated STAT1 (#9167), Erk (#4370), p38 (#9215), AKT (#9275) and mTOR (#2971), which were purchased from Cell Signaling, rabbit antibodies against the heat shock protein 60 of Chlamydia (obtained by the lab), and mouse anti-human β-actin (Sigma, #A5441).

Techniques: Protein-Protein interactions, Incubation, Activation Assay, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Phospho-proteomics

Journal: JCI Insight

Article Title: JAK inhibitor blocks COVID-19 cytokine–induced JAK/STAT/APOL1 signaling in glomerular cells and podocytopathy in human kidney organoids

doi: 10.1172/jci.insight.157432

Figure Lengend Snippet: JAK/STAT signaling mediates COVID-19 cytokine–induced APOL1 expression. ( A ) Experimental design: Primary human podocytes isolated from donor kidney and primary GECs were cultured with or without component cytokines to determine the effect on cellular APOL1 expression. ( B ) Positive immunofluorescence staining of podocytes for Wilms tumor 1 (podocyte marker). Additional marker staining information is given in . Original magnification, 20×. ( C ) Quantitative PCR (qPCR) analysis of GECs compared with HEK cells showing enrichment in PECAM1 gene expression (an endothelial marker, also known as CD31). Data are expressed as mean ± SD; n = 3. Significance difference assessed by 2-tailed t test, with significance set at P < 0.05. ( D ) qPCR analysis of APOL1 mRNA transcript level in GECs and ( E ) podocytes treated for 48 hours with specified individual cytokines, combination of cytokines, and combination of cytokines plus a JAK inhibitor (baricitinib). GAPDH was used for normalization. All cytokine concentrations were 50 ng/mL. Cytokine conditions inducing a >1.5-fold APOL1 transcript compared with media-treated control were further analyzed for significance using unpaired t test with Holm-Šidák correction for multiple comparisons. P values reported are the adjusted P values. Significance was set at P < 0.05. Data are expressed as mean ± SD; n = 6 for podocyte control and n = 3 for all others. Cytokine conditions inducing significantly different APOL1 expression compared with control are indicated in red. ( F ) WB analysis of APOL1, phosphorylated STAT1–3, and total STAT1–3 in GECs after 48-hour treatment with indicated cytokines, combination of cytokines, and combination of cytokines plus a JAK inhibitor (baricitinib). GAPDH was used as housekeeping protein for comparator. ( G ) WB analysis of APOL1 in podocytes after 48-hour treatment with individual cytokines, combination of cytokines, and combination of cytokines plus baricitinib. Vinculin was used as the housekeeping protein for a comparator. NEPH1 and WT1 are podocyte markers.

Article Snippet: Primary Abs against the following proteins were used: APOL1 rabbit anti-human (Genentech, 3.1C1 and 3.7D6; Western blot [WB] 1:5000 [final concentration 0.05 μg/mL; Genentech, 5.17D12]; IHC 1:4000 [final concentration 0.95 μg/mL], per ref. ); APOL1 mouse anti-human (Genentech, 4.17A5; immunofluorescence [IF] 1:2000 [final concentration 2.13 μg/mL]); GAPDH mouse anti-human (Santa Cruz Biotechnology, sc47724; WB 1:200); vinculin mouse anti-human (MilliporeSigma, V9131; WB 1:200); NEPH1 mouse anti-human (Santa Cruz Biotechnology, sc373787; WB 1:300); WT1 rabbit anti-human (Abcam, ab89901; WB 1:1000); STAT1 mouse anti-human (Cell Signaling Technology [CST], 9176s; WB 1:1000); STAT2 rabbit anti-human (CST, 72604; WB 1:1000); STAT3 mouse anti-human (CST, 9139; WB 1:1000); phosphorylated STAT1 (Y701) rabbit anti-human (CST, 9167; WB 1:1000); phosphorylated STAT2 (Y690) rabbit anti-human (CST, 88410; WB 1:1000); phosphorylated STAT3 (Y705) rabbit anti-human (CST, 9145; WB 1:2000); PODXL goat anti-human (R&D Systems, AF1658; IF 1:500), E-cadherin rabbit anti-human (CST, 3195S; IF 1:200), and NEPH1 mouse anti-human (Santa Cruz Biotechnology, sc-373787; IF 1:100).

Techniques: Expressing, Isolation, Cell Culture, Immunofluorescence, Staining, Wilms Tumor Assay, Marker, Real-time Polymerase Chain Reaction, Gene Expression, Control

Journal: Journal of Immunology Research

Article Title: Respiratory Syncytial Virus Nonstructural Proteins Upregulate SOCS1 and SOCS3 in the Different Manner from Endogenous IFN Signaling

doi: 10.1155/2015/738547

Figure Lengend Snippet: Expression of RSV NS proteins resulted in impaired IFN- α -induced STAT phosphorylation. A549 cells were incubated with or without IFN- α (5000 U/mL) for 30 min and then transfected with empty vectors (a), pNS1 (b), or pNS2 (c) for the indicated times. Total protein lysates were subjected to Western blot analysis using the following antibodies: anti-pSTAT1 (1 : 1000), anti-STAT1 (1 : 1000), anti-pSTAT2 (1 : 1000), or anti-STAT2 (1 : 1000). The results shown are representative of three independent experiments. The data were analyzed by densitometry, normalized to β -actin protein levels, and are shown as the mean ± SE. ∗ p < 0.05 versus untreated controls.

Article Snippet: The primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) as follows: rabbit anti-human STAT1pAb; rabbit anti-human STAT2 pAb; rabbit anti-human phosphorylated STAT1 pAb; rabbit anti-human phosphorylated pSTAT2 pAb; rabbit anti-human SOCS1 pAb; rabbit anti-human SOCS3 pAb; rabbit anti-Flag mAb; and rabbit anti-HA mAb.

Techniques: Expressing, Phospho-proteomics, Incubation, Transfection, Western Blot