The samples were obtained from extraorbital lacrimal glands, which were removed bilaterally for histopathological evaluations and immunoblotting 2, 6, 8, and 12 weeks after irradiation. The tissues were homogenized in RIPA buffer (#89900. Thermo scientific. Waltham, MA, USA). Amounts of protein were measured by BCA assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Proteins (50 µg) were loaded and electroblotted. The blots were probed with primary antibodies against monoclonal anti-NFAT5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal anti-cleaved caspase-3, anti-phosphorylated NF-κB, p38, pJNK, and pERK purchased from Cell Signaling Technology (Danvers, MA, USA) at 4 °C overnight. The primary antibody was visualized by a secondary antibody and an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Anti phosphorylated nf κb
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-phosphorylated NF-κB is a primary antibody that specifically detects the phosphorylated form of the NF-κB transcription factor. NF-κB is a key regulator of cellular processes and its phosphorylation is a crucial step in its activation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ecl kit, by Cytiva (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Anti np protein, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit cy3 conjugate secondary antibody, by Merck Group (1 mentions)
Dm5000 upright microscope, by Leica (1 mentions)
Lab tek 2 chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Ab76429, by Abcam (1 mentions)
Ab92700, by Abcam (1 mentions)
Anti α tubulin, by GeneTex (1 mentions)
Anti iκbα, by Abcam (1 mentions)
Gtx127375, by GeneTex (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
Gtx628802, by GeneTex (1 mentions)
Azithromycin, by Selleck Chemicals (1 mentions)
Bez235, by Selleck Chemicals (1 mentions)
Anti phosphorylated c jun, by Cell Signaling Technology (1 mentions)
Anti phosphorylated c fos, by Cell Signaling Technology (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
5 protocols using anti phosphorylated nf κb
1
Irradiation-Induced Lacrimal Gland Pathways
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After drug treatment, the cell lysate was separated by SDS-PAGE and transferred to nitrocellulose membrane. After being blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (v/v) and 5% BSA (w/v) at room temperature for 2 h, the membranes were rinsed and incubated at 4 °C overnight with anti-NP protein (Santa Cruz, USA), anti-phosphorylated NF-κB, Akt, EGFR, PKCα antibodies, or anti-β-actin and GAPDH antibodies (Cell Signaling Technology, Danvers, USA) as control. The membranes were washed and incubated with AP-labeled secondary antibody (1:2000 dilutions) at RT for 2 h. The protein bands were then visualized by incubating with the developing solution (p-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP)) at RT for 30 min. The relative densities of proteins were all determined by using ImageJ (NIH) v.1.33 u (USA).
3
Phosphorylated NF-κB Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
106/ml LAN5 cells were cultured on Lab-Tek II Chambered Coverglass (Nunc) and treated as described above. After washing in PBS, the cells were fixed in 4% paraformaldehyde for 30 min and stored at 4°C. After incubation with 3% BSA/PBS for 1 h, the cells were immunostained with anti-phosphorylated-NFκB (1 : 100; Cell Signaling) antibody at 4°C overnight. After washing in PBS, the samples were incubated with anti-rabbit Cy3-conjugate secondary antibody (1 : 500; Sigma). For nuclear staining, the cells were incubated with Hoechst 33258 (5 μg/ml) for 20 minutes. After washing, the cells were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20x magnification.
4
Immunoblot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot assays were conducted as described previously [28 (link)]. The following primary antibodies were used: anti-IGFBP3 (1:1000, GTX113364, GeneTex, Irvine, CA, USA), anti-cytochrome c (1:1000, 556,433, BD Biosciences, Franklin Lakes, NJ, USA), anti-cleaved caspase 3 (1:500, IMG-144A, IMGENEX, San Diego, CA, USA), anti-light chain 3B (1:500, LC3B, GTX127375, GeneTex), anti-NF-κB (1:1000, #6956, Cell Signaling), anti-phosphorylated NF-κB (1:1000, #3033, Cell Signaling), anti-IκBα (1:1000, ab76429, Abcam, Cambridge, UK), anti-phosphorylated IκBα S32 (1:1000, ab92700, Abcam), and anti-α-tubulin (1:5000, GTX628802, GeneTex). Protein levels were determined by measuring the intensity of bands on western blots using ImageJ (National Institutes of Health, Bethesda, MD, USA). Protein levels were normalized against α-tubulin as an internal control. The relative ratio was calculated by dividing the normalized protein levels in stably expressing cells with that in control cells.
5
Inhibition of NF-κB and MAPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following reagents were used: dexamethasone (Sigma, Dorset, UK), H2O2 (Sigma, Dorset, UK), azithromycin, LY294002, BEZ235 (Selleck, Houston, USA), and tumor necrosis factor α (TNFα) (Sigma, Dorset, UK). The following antibodies were used: goat anti-rabbit (Abcam, Cambridge, UK), anti-phosphorylated NF-κB (Cell Signaling Technology, Danvers, USA), anti-phosphorylated c-Jun (Cell Signaling Technology, Danvers, USA), and anti-phosphorylated c-Fos (Cell Signaling Technology, Danvers, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!