mouse α stat1  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: A non-immunological role for γ-interferon–inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption

doi: 10.1126/sciadv.abd3684

Figure Lengend Snippet: ( A ) mRNA expression of the osteoclast markers cathepsin K and tartrate-resistant acid phosphatase (TRAP), as well as GILT in M-CSF–stimulated WT bone marrow cells treated with or without RANKL as measured by quantitative polymerase chain reaction (qPCR) ( n = 8 to 9). Expression was normalized to 18 S ribosomal RNA and made relative to M-CSF control samples. ( B ) Representative Western blot image depicting GILT protein levels following lysis of WT BMMØs (−RANKL) or osteoclasts (+RANKL). Mature GILT expression was normalized to total protein levels ( n = 5). ( C ) Detection of GILT (green) and cathepsin K (red) by immunofluorescence microscopy in BMMØs (M-CSF) or osteoclasts (M-CSF + RANKL). Scale bars, 50 μm. Colocalization of the GILT and cathepsin K signals was assessed by calculating the Pearson’s correlation coefficient in BMMØs (0.743 ± 0.04) and osteoclasts (0.703 ± 0.07) ( n = 3 to 4 images). ( D ) STAT1 phosphorylation status within WT osteoclast precursors left untreated (−) or treated for 15 min (+) with RANKL (200 ng/ml) or IFN-γ (100 U/ml). Levels of phosphorylation were normalized to total STAT1 levels and made relative to untreated controls ( n = 3). ( E ) GILT protein expression by WT or STAT1 −/− osteoclast precursors following 48-hour treatment with M-CSF + RANKL (15 ng/ml + 100 ng/ml) or M-CSF only. Mature GILT levels were normalized to total protein levels and made relative to M-CSF–only controls ( n = 3). (A to C) Cells were differentiated for 6 days with M-CSF (15 ng/ml) and RANKL (100 ng/ml), or M-CSF (15 ng/ml) only, throughout the entirety of each experiment. (D and E) Precursor cells were expanded in M-CSF (15 ng/ml) for 48 hours before described treatments. (A to E) Error bars are presented as means ± SEM. * P < 0.05, ** P < 0.01 by paired (A to D) or unpaired (E) Student’s t test.

Article Snippet: Rabbit α-mouse P-S727 STAT1 (#9177), P-Y701 STAT1 (#9167), and total STAT1 (#9172) antibodies (all Cell Signaling Technologies) were used to evaluate STAT1 signaling and were probed using a goat α-rabbit IgG-HRP secondary antibody (#7074, Cell Signaling Technology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Lysis, Immunofluorescence, Microscopy, Phospho-proteomics

Journal: Journal of neurovirology

Article Title: Early induction of interferon-responsive mRNAs in Creutzfeldt-Jakob disease

doi:

Figure Lengend Snippet: Activation of kinases and transcription factors in CJD microglia. Representative western blots for IRF-3 (A), total and phosphorylated forms of the p44 and p42 MAP kinases (B), as well as phosphorylated STAT1 and total STAT1α (C). The phospho-STAT1 antibody recognizes both the STAT1α (p91) and STAT1β (p84) forms of the molecule, which are generated by alternative splicing. Normalization to α-tubulin protein was used to control for differences in protein loading on the blots. Equivalent levels of total p44/p42 MAP kinases and STAT1α in normal and CJD microglia confirm that any changes in the phosphospecific forms of these molecules are indeed the result of differential phosphorylation between these cell populations.

Article Snippet: Mouse antibodies against STAT1 α (1:500) and phospho-STAT1 (1:500) were purchased from Zymed (South San Francisco, CA), as was a rabbit antibody against IRF-3 (1:500).

Techniques: Activation Assay, Western Blot, Generated

Journal: Journal of neurovirology

Article Title: Early induction of interferon-responsive mRNAs in Creutzfeldt-Jakob disease

doi:

Figure Lengend Snippet: Comparison of typical innate immune signaling cascades (top panel) with those activated by CJD infection (bottom panel). The pathway diagram has been adapted from Taniguchi and Takaoka (2002). Dotted lines indicate interactions that are indirect or hypothetical. Typical innate immune responses are initiated by recognition of certain foreign protein motifs, mediated by toll-like receptors (TLRs) or other molecules (at A). This leads to the activation of NFκB and IRF-3 transcription factors, which induce the expression of interferons, RANTES, ISG15, CXCL10, and other genes as part of a primary response (at B). Newly synthesized interferons (at C), acting through the type I interferon receptor (IFNAR1/2), activate the STAT1/STAT2/IRF-9 transcription factor complex and instigate two major effects. First, this complex amplifies the expression of some primary response genes (at D). Second, this complex stimulates the expression of a distinct set of transcripts, termed secondary response genes (at E). One of these secondary response genes, the IRF-7 transcription factor, augments transcription of type I interferons and thus completes a positive feedback loop in the innate immune response (at F). In comparison to nonspecific stimuli, the CJD agent does not appear to follow the same recognition pathway (G) leading to increased interferon transcription (H) or synthesis (I). The CJD agent instead elevates certain primary (J) and secondary (K) response genes in an interferon-independent manner, which may involve IRF family transcription factors. The induction of IRF-7 in CJD microglia in principle should result in positive feedback onto the IFNβ promoter (L), priming the cells to exhibit a potentiated response to a challenge stimulus such as poly I:C.

Article Snippet: Mouse antibodies against STAT1 α (1:500) and phospho-STAT1 (1:500) were purchased from Zymed (South San Francisco, CA), as was a rabbit antibody against IRF-3 (1:500).

Techniques: Infection, Activation Assay, Expressing, Synthesized

Journal: Journal of neurovirology

Article Title: Early induction of interferon-responsive mRNAs in Creutzfeldt-Jakob disease

doi:

Figure Lengend Snippet: PCR conditions

Article Snippet: Mouse antibodies against STAT1 α (1:500) and phospho-STAT1 (1:500) were purchased from Zymed (South San Francisco, CA), as was a rabbit antibody against IRF-3 (1:500).

Techniques: