Control (shCo) and ERK3 knockdown (shERK3) HT-29 cells were seeded on coverslips. Next day, medium was exchanged to FBS-free medium and cells were stimulated with LPS (200 ng/ml) for 1.5 hr. After the treatment, cells were fixed in 3.7% formaldehyde (Cat# CP10.1, Roth) for 15 min, followed by washing with PBS and 3 min permeabilization using 0.1% Triton X-100 (AppliChem). After washing twice with PBS, cells were blocked with 1% BSA (Sigma) in PBS for 15 min and washed once with PBS. Staining was performed with anti-ERK3 antibody (Cat# MAB3196, R and D) and anti-c-Jun (60A8) (Cat# 9165, Cell Signaling) (dilution 1:400) antibody in blocking solution for 1 hr at RT. Afterwards, cells were washed with PBS and incubated with secondary antibodies: anti-rabbit IgG-Alexa 488 (Cat# A11008, ThermoFisher), secondary anti-mouse IgG-Cyanine3 (Cat# A10521, Thermo Fisher Scientific) at 5 µg/ml and DNA dye (Hoechst 33342) (Cat# H3570) at 10 µg/ml in blocking solution for 1 hr at RT in the dark. Samples were washed twice with PBS and cells were mounted onto glass slides using Moviol (+DABCO) (Sigma). Cells were imaged using a Leica DMi8 confocal microscope (63x, oil immersion objective).
Anti c jun 60a8
Manufactured by Cell Signaling Technology
Anti-c-Jun (60A8) is a rabbit monoclonal antibody that recognizes the c-Jun protein. C-Jun is a transcription factor that is a subunit of the AP-1 complex and plays a role in regulating gene expression in response to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Dna dye hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Dmi8 confocal microscope, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by AppliChem (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti flag m2, by Cell Signaling Technology (1 mentions)
Anti fra1 r 20, by Santa Cruz Biotechnology (1 mentions)
Anti c fos, by Cell Signaling Technology (1 mentions)
Anti smad4, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Apoptosis detection kit, by Abcam (1 mentions)
Ast alt assay kit, by Nanjing Jiancheng (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Meta imaging series, by Molecular Devices (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti irf3, by Active Motif (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
6 protocols using anti c jun 60a8
1
Immunofluorescence Assay for ERK3 and c-Jun
2
Immunohistochemical Analysis of Sciatic Nerve
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sciatic nerves were isolated and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. After washing with PBS and 30% sucrose, nerves were embedded in OCT and frozen at −80 °C. Cryo-sections were acquired in cross-section orientation at 15 μm thickness. Slices were brought to room temperature and then incubated with blocking solution (2% bovine serum albumin, 2% normal goat serum, 0.2% Triton in 1× PBS). The following primary antibodies were diluted in blocking solution and incubated for 2 h at room temperature: Anti-S100 beta [EP1576Y] (Alexa Fluor 488—Abcam 1:200) and Anti-c-Jun (60A8, Cell Signaling 1:200). Samples were then washed three times (5 min each) in 1× PBS, and slides were incubated with secondary species-specific Invitrogen antibodies for 1 h or mounted using Vectashield with DAPI (Vector Labs) to label nuclei. Fluorescent images were obtained with a Zeiss AxioImager microscope. CZI files were analyzed using FIJI. All data were quantified blindly. N = 3 per genotype. c-Jun data are shown in Fig. 6 .
3
Immunoprecipitation and Immunoblotting for p73 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared in lysis buffer containing 0.5% Nonidet P-40 as described (36 (link)). For immunoprecipitation, 0.5–1.0 mg of lysate was used with agarose-immobilized anti-FLAG M2 antibody (Stratagene). Bead-bound proteins were isolated by boiling in 2× SDS sample buffer followed by separation on SDS-polyacrylamide gels. Immunoblotting was performed with the following antibodies: anti-p73 (ER15 and GC15, Oncogene), anti-FLAG M2, anti-phosphorylated c-Jun Ser-63 (9261, Cell Signaling Technology), anti-c-Jun (60A8, Cell Signaling Technology), anti-Fra1 (R-20, Santa Cruz Biotechnology), and anti-actin (Sigma).
4
Western Blotting of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting were performed according to ref. 102 (link), using the following antibodies: anti-cFos (2250, Cell Signaling. Dilution 1/1000), anti-cJun (60A8; 9165, Cell Signaling. Dilution 1/1000), anti-alfa-Tubulin (DM1A; T9026, Sigma. Dilution 1/1000), anti-Erk1/2 (P28482, Millipore. Dilution 1/1000), and anti-Phospho-Erk1/2 (thr202/Tyr204) (9101, Cell Signaling. Dilution 1/1000), anti-SMAD4 (SC-7996, Santa Cruz. Dilution 1/250). As secondary antibodies anti-mouse-HRP and anti-rabbit-HRP (Sigma-Aldrich. Dilution 1/1000) were used.
5
Liver Histopathological and Functional Analyses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues (all lobes) were fixed in 10% formalin overnight, and then dehydrated and embedded in paraffin blocks, which were sectioned at a thickness of 5 μm. For Sirius red staining, sections were stained with hematoxylin (Weigert’s) for 8 min, washed with running tap water for 10 min, followed by incubation with 0.1% (wt/vol) Sirius red diluted in picric acid solution for 1 h. The slides were then rinsed in two quick changes of 0.5% (vol/vol) acetic acid to remove unbound dye. For immunostaining, sections were first heated in sodium citrate buffer to retrieve antigen. Sections were then blocked in 10% FBS blocking buffer followed by incubation with the following primary antibodies: anti-c-Jun (60A8; Cell Signaling), anti-F4/80 (SP115; Thermo Fisher Scientific), and anti-Ki67 (15580; Abcam), before quenching endogenous peroxidase using 3% hydrogen peroxide. TUNEL staining was performed as per manufacturer’s recommendations using the apoptosis detection kit (206386; Abcam). To quantify staining, 20 randomly taken images of 10× fields per section were evaluated by Meta Imaging Series (Molecular Devices) software.
A small portion of the liver was homogenized in ice cold NaCl buffer. The supernatant was collected, normalized before subjected to AST/ALT measurements using an AST/ALT Assay kit (Nanjing Jiancheng Bioengineering Institute) according to the supplier’s protocol.
A small portion of the liver was homogenized in ice cold NaCl buffer. The supernatant was collected, normalized before subjected to AST/ALT measurements using an AST/ALT Assay kit (Nanjing Jiancheng Bioengineering Institute) according to the supplier’s protocol.
6
Immunoblot Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedures used for preparation of Whole Cell Extracts (WCE), resolution by SDS-PAGE electrophoresis and immunoblot was fully detailed in93 . The following primary antibodies were used in this study: anti-MKP-1/DUSP1 (M18, #sc-1102), anti-α-tubulin (B-7, #sc-5286) and anti-NFκB p65 (C-20, #sc-372) were obtained from Santa Cruz Biotechnology. Anti-phosphoT183/Y185 SAPK/JNK (G9, #9255), anti-SAPK/JNK (#9252), anti-phosphoT180/Y182 p38 MAPK (D3F9, #4511), anti-p38 MAPK (D13E1, #8690), anti-phosphoS536 NF-kappaB p65 (93H1, #3033), anti-phosphoS32 IκBα (14D4, #2859), anti-IκBα (#9242), anti-phosphoT71 ATF-2 (11G2, #5112), anti-ATF-2 (20F1, #9226), anti-phosphoS73 c-Jun (D47G9, #3270), anti-c-Jun (60A8, #9165), anti-cleaved Caspase 9 (D315, #9505) and anti-cleaved Caspase 3 (D175, #9664) were from Cell Signaling. Anti-actin clone AC-15 (#A5441) and anti-Flag M2 (#F1804) were purchased from Sigma-Aldrich, anti-IRF3 (#39033) was from Active Motif and anti-RSV (#AB1128) was from Chemicon International. Anti-phosphoS396 IRF3 was described in38 (link) and anti-SeV was obtained from Dr. J. Hiscott, McGill University, Montreal, Canada. HRP-conjugated goat anti-rabbit and rabbit anti-goat (Jackson Laboratories), and goat anti-mouse (Kirkegaard & Perry Laboratories) were used as secondary antibodies. Immunoblots were quantified using the ImageQuantTL software (Molecular Dynamics).
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!