cfos and cjun antibodies  (Active Motif)

Journal: Applied Microbiology and Biotechnology

Article Title: Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines

doi: 10.1007/s00253-022-12321-2

Figure Lengend Snippet: A Relative expression profile of cell signaling molecules at mRNA level in EAEC-T8–infected target ST-silenced respective cell types. The bar diagrams indicate EAEC-T8–induced expression of ERK1, ERK2, JNK, p38, NFκB, cJun, cFos, STAT3, and IL-8 in ST6GAL-1 and ST6GAL-2–silenced HCT-15 and INT-407 cells, respectively, as compared to respective negative control and experimental control. One-way ANOVA, followed by Tukey’s multiple comparison test, was used; *** p < 0.001, ** p < 0.01, and * p < 0.05 indicate the level of significance; ns, non-significant. B Expression profile of cell signaling molecules at protein level in cells. Western blots showing the expression of a MAPKs [pERK1/2, ERK1, ERK2, pJNK, JNK, pp38, and p38] and b transcription factors [IκB, NF-κB (cytoplasmic), NF-κB (nuclear), cFos (nuclear), cJun (nuclear), pSTAT-3 (nuclear) and STAT-3 (nuclear)] in ST6GAL-1 and ST6GAL-2–silenced respective HCT-15 and INT-407 cell types as compared to the respective negative control and experimental control. In the case of MAPKs, the blots were probed with anti-pERK, anti-pJNK, and anti-pp38 as the primary antibodies followed by incubation with HRP-conjugated secondary antibody. The bands on the blots were then stripped off and reprobed with the antibody to total ERK2 and ERK1, JNK, and p38. For transcription factors, the blots were probed with NF-κB (cytoplasmic), NF-κB (nuclear), cFos, cJun, and pSTAT-3 (nuclear) as the primary antibodies followed by incubation with HRP-conjugated secondary antibody. The NF-κB (cytoplasmic) and pSTAT-3 (nuclear) bands on the blots were then stripped off and reprobed with the antibody to IκB and STAT-3. Normalization was done by using β-actin (internal control). C1, lipofectamine-treated; C2, esi-FLUC-RNA–transfected; T, esi-ST6GAL-1-RNA/esi-ST6GAL-2-RNA–transfected EAEC-T8–infected HCT-15 and INT-407 cells; M r , protein markers. C Bar diagrams revealing the expression of a MAPKs and b transcription factors in EAEC-T8–infected ST6GAL-1 and ST6GAL-2–transfected respective cell line as evaluated by Scion analysis with ImageJ software. The intensity of the band of each parameter was normalized to that of β-actin as the internal control. Further, the level of expression of each activated MAPK was obtained by normalization of each value against the value of respective total MAPK. The expression of each molecule in lipofectamine-treated control cells (C1) was set to 1, with respect to which the expression of the respective molecule in esi-FLUC RNA–transfected cells (C2) and esi-ST6GAL-1-RNA and esi-ST6GAL-2-RNA–transfected cells (T) was calculated. D ELISA-based estimation of IL-8 secretion by EAEC-T8–infected target ST-silenced respective cell types as compared to that of response in control cells (lipofectamine-treated cells and esi-FLUC RNA–transfected cells). One-way ANOVA, followed by Tukey’s multiple comparison test, was applied which revealed a significant reduction in secretory IL-8 concentration in each of the ST-silenced cell line [ST6GAL-1–silenced HCT-15 cells, 1451.7 pg/ml; ST6GAL-2–silenced INT-407 cells, 1389.1 pg/ml] in comparison to the respective vehicle control [HCT-15, 2269.583 pg/ml; INT-407, 2230 pg/ml); *** p < 0.001 (esi-ST6GAL-1-RNA and esi-ST6GAL-2-RNA vs. the lipofectamine-treated cells); ns, non-significant (esi-FLUC-RNA vs. the lipofectamine-treated cells)

Article Snippet: After blocking, membranes containing the cytoplasmic proteins were incubated (overnight, 4 °C) separately with the specific antibody (diluted in 2.5% SM-TBST 0.1% ) against NF-κB and total STAT-3 (Santa Cruz Biotechnology), while the membranes containing the nuclear proteins were incubated (overnight, 4 °C) separately with the specific antibody against NF-κB, pSTAT-3, cFos, and cJun (Santa Cruz Biotechnology, USA; Table ).

Techniques: Expressing, Infection, Negative Control, Western Blot, Incubation, Transfection, Software, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Cfos and cjun mutations detected in synovial tissue of rheumatoid arthritis (RA) and osteoarthritis (OA) patients.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques:

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Effects of cjun and cfos mutations on reporter gene expression. The graph shows pentameric AP-1 site- and matrix metalloproteinase 1 (MMP-1) promoter-dependent expression of firefly luciferase 2 days following transfection of NIH-3T3 cells with wt ( A ) or mutated ( B , C ) cfos and cjun expression plasmids (determined in biological triplicates; means ± standard error of the mean). Firefly luciferase expression levels were normalized to renilla luciferase expression levels in the respective samples (transfection and normalization control). Results are presented as relative luciferase activity (in x-fold) related to the expression level in the presence of the control vector ( A ), cfos wt expression vector ( B ), or cfos and cjun wt vectors ( C ). For normalization and to correct for different transfection efficiencies, the renilla luciferase-expressing vector pRL-CMV was co-transfected. In all analyzed samples, luciferase expression was easy to detect and clearly exceeded background levels in non-transfected cells. * p ≤ 0.05 vs. controls, i.e., pCMX ( A ), fosWt ( B ), and fosWt/junWt ( C ); +, °, §, ‡, and # p ≤ 0.05 vs. respective other mutants; fos73 represents the double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Gene Expression, Expressing, Luciferase, Transfection, Control, Activity Assay, Plasmid Preparation, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: DNA binding activity of AP-1 complexes in transfected K4IM fibroblasts and protein expression of cfos and cjun variants. ( A ) Electrophoretic mobility shift assay (EMSA) of nuclear extracts from transfected K4IM cells. The phosphor-image exemplarily shows binding of radio-labelled AP-1 sites by AP-1 complexes formed in K4IM cells transfected with the indicated cfos and/or cjun variants ( n = 1). ( B ) In the corresponding whole cell extracts, protein amounts of cFos and cJun following transfection were detected by Western Blot ( n = 1). Loading control: glyceraldehyde 3-phosphate dehydrogenase (GAPDH); fos73: double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Binding Assay, Activity Assay, Transfection, Expressing, Electrophoretic Mobility Shift Assay, Western Blot, Control, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Quantitation of MMP-1 mRNA expression in jun-/fos-transfected primary human fibroblast-like synoviocytes (FLS). ( A – C ) The graph shows the MMP-1 mRNA expression (detected by qPCR) in primary FLS derived from RA and OA patients (without detectable genetic variations in the jun and fos genes; n = 3 each, mean ± SD) following transfection of cjun and/or cfos expression vectors. ( A ) MMP-1 expression in FLS transfected with the indicated cfos variants. ( B ) MMP-1 expression in FLS transfected with the indicated cjun variants. ( C ) MMP-1 expression in FLS transfected with combinations of cfos and cjun variants. MMP-1 mRNA levels in fosWt- ( A ), junWt- ( B ), or fosWt/junWt-transfected cells ( C ) were set as 1. Statistical analyses: Mann–Whitney U-test, * p ≤ 0.05 vs. the respective controls; fos73: double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Quantitation Assay, Expressing, Transfection, Derivative Assay, MANN-WHITNEY, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Quantitation of IL-6 mRNA expression in jun-/fos-transfected primary human FLS. ( A – C ) The graph shows the IL-6 mRNA expression (detected by qPCR) in primary FLS derived from RA and OA patients (without detectable genetic variations in the jun and fos genes; n = 3 each, mean ± SD) following transfection of cjun and/or cfos expression vectors. ( A ) IL-6 expression in FLS transfected with the indicated cfos variants. ( B ) IL-6 expression in FLS transfected with the indicated cjun variants. ( C ) IL-6 expression in FLS transfected with combinations of cfos and cjun variants. IL-6 mRNA levels in fosWt- ( A ), junWt- ( B ), or fosWt/junWt-transfected cells ( C ) were set as 1. Statistical analyses: Mann–Whitney U-test, * p ≤ 0.05 vs. the respective controls; fos73: double mutant fos73/fos125.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques: Quantitation Assay, Expressing, Transfection, Derivative Assay, MANN-WHITNEY, Mutagenesis

Journal: Life

Article Title: Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue

doi: 10.3390/life11010005

Figure Lengend Snippet: Frequency analysis of mutations in GnomAD.

Article Snippet: For cFos and cJun protein detection, SDS-PAGE (10%) was performed using 10 μL of each whole cell extract sample, followed by Western Blot analysis using the respective cFos and cJun antibodies from the TransAM AP-1 Family Kit (Active Motif, Rixensart, Belgium).

Techniques:

Journal: Toxicology letters

Article Title: Nitrogen mustard exposure of murine skin induces DNA damage, oxidative stress and activation of MAPK/Akt-AP1 pathway leading to induction of inflammatory and proteolytic mediators

doi: 10.1016/j.toxlet.2015.04.006

Figure Lengend Snippet: Mice were exposed topically to either 3.2 mg NM in acetone or acetone alone and dorsal skin was collected at 12, 24, 72 and 120 h time points after exposure. Skin tissue lysates were prepared and nuclear lysates were subjected to DNA binding activity by EMSA (A), and western immunoblotting (B and C) as detailed under ‘Materials and Methods’ Section 2. EMSA was carried out using AP1 oligo (A), and western blot analysis was done for phospho- and total cFos and cJun (B and C). Protein loading was checked by stripping and re-probing the membranes with β-actin antibody. Western blot results were quantified by densitometric analysis of representative immunoblots for phospho-cFos total cFos phospho-cJun and total cJun. Results shown are representative of 2–3 animals in the study group.

Article Snippet: Phosphor-ylated anti-cJun anti-cFos-Rad 51, non-phosphorylated anti-cJun anti-cFos antibodies and consensus sequences of double stranded AP1 oligonucleotides were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Binding Assay, Activity Assay, Western Blot, Stripping Membranes

Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Stress Activates the Hepatic Activator Protein 1 Complex via Mitogen Activated Protein Kinase-Dependent Signaling Pathways

doi: 10.1371/journal.pone.0103828

Figure Lengend Snippet: HepG2 cells pretreated with MEK1 inhibitor (PD) or vehicle followed by treatment with tunicamycin (Tm) or vehicle (control) for 6 hours. A) Representative Western blot of total and phosphorylated ERK, JNK, CFOS, and CJUN. Relative mRNA expression of B) GRP78/BIP , C) CHOP , D) CFOS , E) FRA-1 , F) CJUN , and G) JUND . Mean (n = 6) ± SD. * p<0.05.

Article Snippet: Protein detection was performed using polyclonal rabbit antibodies to total and phosphorylated cFos, cJun, ERK, and JNK (Cell Signaling Technology, Danvers, MA).

Techniques: Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Stress Activates the Hepatic Activator Protein 1 Complex via Mitogen Activated Protein Kinase-Dependent Signaling Pathways

doi: 10.1371/journal.pone.0103828

Figure Lengend Snippet: HepG2 cells pretreated with JNK inhibitor (SP600125) or vehicle followed by treatment with tunicamycin or vehicle (control) for 6 hours. A) Representative Western blot of total and phosphorylated JNK, ERK, CJUN, and CFOS. Relative mRNA expression of B) GRP78/BIP , C) CHOP , D) CJUN , E) JUND , F) CFOS , and G) FRA-1 . Mean (n = 6) ± SD. * p<0.05.

Article Snippet: Protein detection was performed using polyclonal rabbit antibodies to total and phosphorylated cFos, cJun, ERK, and JNK (Cell Signaling Technology, Danvers, MA).

Techniques: Control, Western Blot, Expressing