Nuclear and cytosol fractions were prepared using a Nuclear and Cytoplasmic Isolation kit (Thermo Scientific). All cells used for immunocytochemistry were fixed with 4% paraformaldehyde (Sigma) and permeabilized with 0.25% Trition X-100 in phosphate-buffered saline containing 1% bovine serum albumin. Fixed cells were incubated with an anti p50 (NF-κB) (1:500; SC-8414, RRID: AB_628015) antibody (Santa Cruz Biotechnology) at 4 °C overnight. After that, cells were incubated with Alexa 488 goat anti-mouse secondary antibody (Thermo Scientific) at 4 °C overnight. Images were obtained using an LSM-710 confocal microscope (Carl Zeiss, Oberkochen, Germany).
Sc 8414
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-8414 is a laboratory equipment product offered by Santa Cruz Biotechnology. The core function of this product is to facilitate experimental procedures in a laboratory setting. Detailed specifications and intended uses are not provided in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Alexa488 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sc 271430, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 555 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti inos, by Thermo Fisher Scientific (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab75850, by Abcam (1 mentions)
Ab49470, by Abcam (1 mentions)
Myc sc 40, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Merck Group (1 mentions)
β catenin, by Merck Group (1 mentions)
T9026, by Merck Group (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
β catenin, by BD (1 mentions)
Gtx100118, by GeneTex (1 mentions)
Gtx109639, by GeneTex (1 mentions)
Immobilon transfer membrane, by Merck Group (1 mentions)
Biospectrum 810 imaging system, by Analytik Jena (1 mentions)
5 protocols using sc 8414
1
NF-κB p50 Subcellular Localization
2
Multicolor Immunofluorescence Analysis
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hBMSCs, RAW264.7 cells, and human umbilical vein endothelial cells (HUVECs) were fixed in 4% paraformaldehyde solution for 15 min. Fixed cells were washed carefully with a PBS solution three times and were then permeabilized for 15 min with 0.3% Triton X-100 in PBS solution. To reduce the nonspecific background, the cells were blocked with a 5% BSA in PBS solution for 1 h. After blocking, the cells were incubated with primary antibodies overnight at 4 °C. The cells were subsequently rinsed with PBS solution and incubated with secondary antibodies for 1 h at RT. After antibody labeling, cells were washed with PBS solution, and nuclei were stained with Hoechst 34,580. The primary antibodies included anti–NF–κB (1:200; sc-8414, Santa Cruz Biotechnology, CA, USA), anti-Arginase 1 (1:200; sc-271,430, Santa Cruz Biotechnology), and anti-iNOS (1:500; Invitrogen, Thermo Fisher Scientific). The secondary antibodies included Alexa Fluor™ 488 donkey anti-rabbit IgG (H + L) and Alexa Fluor™ 555 donkey anti-mouse IgG (H + L) (A-21206 and A-21422, Invitrogen, Thermo Fisher Scientific).
3
Western Blot Analysis of Key Signaling Proteins
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Cell lysates were prepared and subjected to western analysis as described previously (Williams et al., 1993 (link)) using antibodies to the following: α-tubulin (T9026; Sigma-Aldrich; 1:10,000), ASCL2 (4418; EMD Millipore, Watford, UK; 1:500), β-catenin (9587; Cell Signaling Technology, MA, USA; 1:5000), β-catenin (610153; BD Biosciences, CA, USA; 1:10,000), de-phosphorylated (actively signalling) β-catenin (05-665; EMD Millipore; 1:1000), BCL-3 (ab49470; Abcam, Cambridge, UK; 1:1000), BCL-3 (23959; Proteintech, Manchester, UK; 1:2000), MYC (sc-40; Santa Cruz Biotechnology, TX, USA; 1:500), cyclin D1 (2978; Cell Signaling Technology; 1:1000), lamin A/C (4200236; Sigma; 1:5000), LEF1 (2230; Cell Signaling Technology; 1:1000), LGR5 (ab75850; Abcam; 1:1000), NF-κB1 (p50; sc-8414; Santa Cruz Biotechnology; 1:1000), NF-κB2 (p52;05-361; EMD Millipore; 1:1000) and TCF4 (2569; Cell Signalling Technology; 1:1000).
4
Western Blot Analysis of Cellular Proteins
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The cells were dissolved in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, and protease inhibitor Cocktail Set III (Calbiochem). Fifty micrograms of protein were fractionated via 10% SDS-PAGE and transferred to Immobilon Transfer Membranes (Millipore). Primary antibodies used in this study included those against β-actin (GTX109639; dilution 1:10,000; GeneTex), poly (ADP-ribose) polymerase (PARP) (9542S; dilution 1:1000; Cell Signaling Technology), ACE2 (bs-1004R; dilution 1:1000; Bioss), p65 (SC-8008; dilution 1:1000; Santa Cruz Biotechnology), p50 (SC-8414; dilution 1:1000; Santa Cruz Biotechnology) and GAPDH (GTX100118; dilution 1:1000; GeneTex). HRP-conjugated secondary antibodies recognizing mouse-IgG (7076) or rabbit-IgG (7074) were purchased from Cell Signaling Technology and used at dilutions of 1:5000–10,000. The chemiluminescence signal was captured on a BioSpectrum 810 Imaging System with VisionWorks software (UVP).
5
Localization and Interaction of NF-κB Subunits
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For localization of endogenous p65 in MEFs, WT and GSK-3β-null MEFs were
grown on coverslips for 24 h and stimulated with 2 μg/ml
of TNFα for the indicated periods of time. Cells were fixed,
permeabilized, stained with rabbit monoclonal anti-p65 (1 : 300 dilution)
and analyzed via confocal microscopy as previously described.53 (link) To detect subcellular distribution of GSK-3β,
Panc04.03 cells were transfected with in pCMS4-H1P-eGFP- GSK-3β vectors by
electroporation and grown on coverslips for 48 h before staining with mouse
monoclonal anti-flag M2 antibody (1 : 800). For PLA detection of
p50/p65 interaction in situ, we used the Duolink in situ PLA kit
from Olink Bioscience according to the supplier's instructions. Mouse mAb against
p50 (SC8414, Santa Cruz Biotechnology) and rabbit mAb against p65 (#8242, Cell
Signaling Technology) were used as primary antibodies at dilutions of
1 : 50 and 1 : 300, respectively. The anti-rabbit plus and
anti-mouse minus secondary antibodies were used as PLA probes and Texas red as detection
regent (Supplementary information ).
grown on coverslips for 24 h and stimulated with 2 μg/ml
of TNFα for the indicated periods of time. Cells were fixed,
permeabilized, stained with rabbit monoclonal anti-p65 (1 : 300 dilution)
and analyzed via confocal microscopy as previously described.53 (link) To detect subcellular distribution of GSK-3β,
Panc04.03 cells were transfected with in pCMS4-H1P-eGFP- GSK-3β vectors by
electroporation and grown on coverslips for 48 h before staining with mouse
monoclonal anti-flag M2 antibody (1 : 800). For PLA detection of
p50/p65 interaction in situ, we used the Duolink in situ PLA kit
from Olink Bioscience according to the supplier's instructions. Mouse mAb against
p50 (SC8414, Santa Cruz Biotechnology) and rabbit mAb against p65 (#8242, Cell
Signaling Technology) were used as primary antibodies at dilutions of
1 : 50 and 1 : 300, respectively. The anti-rabbit plus and
anti-mouse minus secondary antibodies were used as PLA probes and Texas red as detection
regent (
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