LPS (from Escherichia coli serotype 055:B5) and poly(I:C) was purchased from Sigma-Aldrich. CpG oligonucleotides were purchased from InvivoGen (San Diego, CA). Recombinant human IFN-α, IFN-β, IFN-γ, GM-CSF, IL-4 and TNF-α were purchased from PeproTech. JAK inhibitor pyridone 6 (P6) was obtained from Sigma-Aldrich. PI3-kinase inhibitor LY294002, extracellular signal-regulated kinase (ERK) inhibitor PD98059 and p38 MAPK inhibitor SB202190 were obtained from Calbiochem (La Jolla, CA). The primary antibodies against STAT3, IFNAR1 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibodies against STAT1, AKT, IRF3 and IRF7 were purchased from Cell Signalling Technology (Beverly, MA). The neutralizing antibody against IFN-α and that against IFN-β were purchased from PeproTech. The primary antibody against ATX was produced by our lab as described previously [33 (link)].
Primary antibodies against stat1
Manufactured by Cell Signaling Technology
Primary antibodies against STAT1 are laboratory reagents used to detect and study the STAT1 (Signal Transducer and Activator of Transcription 1) protein in biological samples. STAT1 is a transcription factor that plays a crucial role in the cellular response to cytokines and other extracellular signals.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human ifn α, by Thermo Fisher Scientific (1 mentions)
Cpg oligonucleotide, by InvivoGen (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Ifn β, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Lps from escherichia coli serotype 055 b5, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
P stat1, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
3 protocols using primary antibodies against stat1
1
Immune Response Modulation Mechanisms
2
Western Blot Analysis of STAT1 and OAS1
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Cells were lysed in lysis buffer (Beyotime Institute of Biotechnology) for 30 min at 4˚C, and centrifuged at 12,000 r/min for 10 min, then the supernatants (containing soluble proteins) were quantified using the UV-Vis spectrophotometer (SSI). The protein samples (30-50 µg/lane) were separated on 12% SDS polyacrylamide gels at 20 mA. The separated proteins were transferred to PVDF membrane (EMD Millipore; 1.5 h at 160 mA using a Bio-Rad mini-transblotter), and immunoblotted at 4˚C for 12 h with primary antibodies against STAT1 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9176), p-STAT1 (1:1,000; Cell Signaling Technology, Inc; cat. no. 9167) and OAS1 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 14498), and incubated with horseradish-peroxidase-conjugated secondary antibody (1:10,000; Beijing Zhongshan Jinqiao Biological Technology Co., Ltd.) at room temperature for 1.5 h. Immunoreactive bands were visualized by enhanced chemiluminescence (Beyotime Institute of Biotechnology). The intensities of bands were normalized to β-actin (1:1,000; Beijing Zhongshan Jinqiao Biological Technology. Co., Ltd.) and analyzed using Image J software (Version 1.6.1 for Windows; National Institutes of Health).
3
Immunofluorescence Analysis of STAT1 and STAT3
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HaCaT cells were seeded in a NuncTM Lab-TekTM Ⅱ Chamber SlideTM System (Cat. No. 154534,
Nunc, Thermo, MA, USA) at 5 × 104 cells/well and treated with 40 μM purpurin
for 1 h. After treatment with 10 ng/mL TNF-α/IFN-γ for 1 h, the cells were fixed in 4%
neutral buffered formalin (Cat. No. BBC 0150, BBC Biochemical, WA, USA) for 10 min at room
temperature and washed 2 times with cold PBS. HaCaT cells were permeabilized with 0.2%
Triton X-100 in cold PBS for 10 min at 4°C, washed 3 times with PBS, and blocked with 3%
bovine serum albumin (BSA) in PBS. Next, the cells were incubated with primary antibodies
against STAT1 (Cat. #8394, Cell Signaling Technology) and STAT3 (Cat. No. #9145, Cell
signaling Technology) at 4°C, overnight. After incubation, HaCaT cells were washed with
PBS 3 times, incubated with anti-rabbit Alexa Fluor 488-conjugated secondary antibody at
4°C for 1 h, and washed with PBS 3 times. Then, the cells were incubated with Hoechst
33342 nucleic acid stain (Cat. H3570, Thermo Fisher Scientific, Inc., MA, USA) for 5 min,
mounted with coverslips, and visualized with an Axio Observer Z1 microscope (ZEISS,
Oberkochen, Germany).
Nunc, Thermo, MA, USA) at 5 × 104 cells/well and treated with 40 μM purpurin
for 1 h. After treatment with 10 ng/mL TNF-α/IFN-γ for 1 h, the cells were fixed in 4%
neutral buffered formalin (Cat. No. BBC 0150, BBC Biochemical, WA, USA) for 10 min at room
temperature and washed 2 times with cold PBS. HaCaT cells were permeabilized with 0.2%
Triton X-100 in cold PBS for 10 min at 4°C, washed 3 times with PBS, and blocked with 3%
bovine serum albumin (BSA) in PBS. Next, the cells were incubated with primary antibodies
against STAT1 (Cat. #8394, Cell Signaling Technology) and STAT3 (Cat. No. #9145, Cell
signaling Technology) at 4°C, overnight. After incubation, HaCaT cells were washed with
PBS 3 times, incubated with anti-rabbit Alexa Fluor 488-conjugated secondary antibody at
4°C for 1 h, and washed with PBS 3 times. Then, the cells were incubated with Hoechst
33342 nucleic acid stain (Cat. H3570, Thermo Fisher Scientific, Inc., MA, USA) for 5 min,
mounted with coverslips, and visualized with an Axio Observer Z1 microscope (ZEISS,
Oberkochen, Germany).
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