primary antibodies against the flag and his tags cell signaling #12,698  (Cell Signaling Technology Inc)

Journal: Biomolecules

Article Title: Pharmacological Inhibition of Astrocytic Transglutaminase 2 Facilitates the Expression of a Neurosupportive Astrocyte Reactive Phenotype in Association with Increased Histone Acetylation

doi: 10.3390/biom14121594

Figure Lengend Snippet: Irreversible inhibition of TG2 with the drug VA4 reduces interaction between TG2 and Zbtb7a. ( a ) Input controls of V5-TG2 and FLAG-Zbtb7a transfected into HEK 293TN cells treated with VA4 or vehicle control (DMSO). ( b ) Immunoprecipitation of V5-TG2 pulls down less FLAG-Zbtb7a in cell lysates that were treated with VA4. In ( a , b ) the position at which molecular weight markers (kDa) migrated is indicated at the left of the immunoblots. ( c ) Quantification of the amount of FLAG-Zbtb7a pulled down normalized to the amount of V5-TG2 immunoprecipitated. Treatment of cells with VA4 resulted in a significant reduction in the Zbtb7a co-immunoprecipitated with TG2 compared to DMSO control. Shown as mean and SEM (n = 5 samples per condition from 4 independent biological replicates, unpaired t -test *** p < 0.001). Original images can be found in .

Article Snippet: After blocking, primary antibodies against FLAG tag (CST 8146S), V5 tag (CST 13202S), GAPDH (Proteintech, Rosemont, IL, USA, 60004-1-Ig), acetylated histone H3 K9 (CST 9649S), or beta tubulin (rabbit polyclonal antibody, Proteintech 10094-1-AP) were added to the blots in fresh blocking buffer and incubated at 4 °C overnight.

Techniques: Inhibition, Transfection, Control, Immunoprecipitation, Molecular Weight, Western Blot

Journal: Journal of Translational Medicine

Article Title: KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

doi: 10.1186/s12967-024-05714-6

Figure Lengend Snippet: KLF14 directly binds to the GPX4 promoter region to inhibit GPX4 transcription. ( A ) Integrative Genomics Viewer (IGV) visualization of the CUT&Tag signals of KLF14 at the GPX4 promoter locus. ( B ) Luciferase reporter assay of GPX4 promoter activity in HEK293 cells with or without KLF14 overexpression. Each experiment was performed with 3 technical repeats and was independently repeated three times. The data are presented as the means ± SDs. p values were calculated by an unpaired t test. ( C ) Promoter truncation assay to locate the KLF14 regulatory region in the GPX4 promoter. (Left) Schematic representation of the truncated GPX4 promoter constructs. (Right) Luciferase values calculated in the reporter assays with the empty vector or KLF14 expression constructs. Each experiment was performed with at least two technical replicates and was independently repeated three times.The data are presented as the means ± SDs. p values were calculated by two-way ANOVA. ( D ) Dual luciferase assay with P1, the P2, P3 and P123 mutants. Each experiment was performed with at least two technical replicates and was independently repeated three times. The data are presented as the means ± SDs. p values were calculated by two-way ANOVA. ( E ) Dual luciferase assay with Del 0–500 bp, 0–1000 bp and 0–1500 bp of GPX4 promoter. Each experiment was performed with at least two technical replicates and was independently repeated three times. The data are presented as the means ± SDs. p values were calculated by two-way ANOVA. ( F ) Western blotting using an anti-Flag antibody was performed to detect the expression of Flag-tagged wild-type KLF14 and its various zinc-finger motif mutants. N = 3 independent repeats. ( G ) Semiquantitative analysis of Flag expression based on WB analysis. The data are presented as the means ± SDs. p values were calculated using one-way ANOVA test. ( H ) Immunofluorescence images of truncated KLF14 proteins in SiHa cells (Red: KLF14; blue: nucleus). The data are presented as the means ± SDs; N = 3 repeats. ( I ) Luciferase reporter assay of GPX4 promoter activity in HEK293 cells with the WT-KLF14 and mutated KLF14 constructs. Each experiment was performed with at least two technical replicates and was independently repeated three times. The data are presented as the means ± SDs. p values were calculated by two-way ANOVA.( J - K ) The effects of truncated KLF14 on cell proliferation were evaluated by RTCA ( J , each experiment was performed with 2 technical repeats and was independently repeated three times. p values were calculated by the Kruskal-Wallis test) and the CCK-8 assay ( K , each experiment was performed with 3 technical repeats and was independently repeated three times. The data are presented as the means ± SDs. p values were calculated by one-way ANOVA test). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies against FLAG (CST, #14793S, 1:1000), KLF14 (Invitrogen, #PA5-23784, 1:1000), GPX4 (CST, #52455S, 1:1000), GAPDH (CST, #5174S, 1:1000), and β-actin (Boster, # BM0627, 1:1000) and secondary antibodies against rabbit IgG (CST, #7074, 1:10000) and mouse IgG (CST, #7076, 1:10000) were used.

Techniques: Luciferase, Reporter Assay, Activity Assay, Over Expression, Construct, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, CCK-8 Assay