primary rabbit antibodies against flag  (Proteintech)

Journal: Cell Communication and Signaling : CCS

Article Title: Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling

doi: 10.1186/s12964-024-01874-6

Figure Lengend Snippet: Engineering and characterization of destabilized monobody variants. a Schematic overview of monobody delivery by the T3SS. Monobodies expressed with a secretion signal are translocated through the T3SS needle, which requires unfolding. After refolding, the delivered monobody can interact with the target protein in the cytoplasm and affect target activity and signaling. b Cartoon representation of monobody AS25 (yellow): Abl-SH2 domain (grey) complex (PDB: 5DC4). Diversified residues of monobody scaffold are shown in green. Y45 (blue), a key residue for target binding was mutated to alanine to obtain a low affinity variant. For the destabilization, the A57 residue (red) was mutated to glycine. c Thermodynamic stability of the AS25 variants assessed by thermal shift assay. Derivative fluorescence of one representative was plotted over temperature. Melting temperatures of triplicates were averaged and are shown as mean ± SD. d-f Isothermal calorimetric titration (ITC) of AS25 (panel d), AS25 A57G (panel e) and AS25 Y45A-A57G (panel f) to Abl-SH2. Upper panels: Raw heat signal; lower panels: Integrated calorimetric data of the area for each peak. The continuous line represents the best fit of the data and the binding parameters K d and stoichiometry (N) are calculated from the fit. A representative measurement ( n = 2) for each monobody is shown. Thermodynamic parameters are listed in Supplementary Tables 2–3. g Secretion assay ( n = 3) showing export of YopE 1-138 -AS25-FLAG-HiBiT variants and native T3SS substrates (the translocator proteins SctA and SctB contributing to formation of a pore in the eukaryotic membrane and the regulatory protein SctW) by Y. enterocolitica . Proteins secreted over 180 min were precipitated and analyzed by SDS-PAGE. Left, Coomassie staining (native substrates indicated on right side); right, Western blot anti-FLAG. Expected size: YopE 1-138 -AS25-FLAG-HiBiT: 28.7 kDa (marked with *). h Immunoblot analysis of YopE 1-138 -AS25-FLAG-HiBiT expression levels in the indicated strains used in panel g

Article Snippet: SDS-PAGE gels were blotted on a AmershamTM Protran® Western Blotting nitrocellulose membrane (0.2 μm) [Cytiva (10600001)] using a Trans-Blot Turbo Transfer System [Bio-Rad (1704150)] with the settings: 1.3 A, 25 V, 7 min. Immunoblots were carried out using primary rabbit antibodies against the FLAG peptide (Rockland (600–401-383), 1:5000) in combination with a secondary goat anti-rabbit antibody conjugated to a peroxidase (Sigma (A8275) 1:10,000) and visualized with Immobilon Forte Western HRP substrate (Merck (WBLUF0500)) on a LAS-4000 Luminescence Image Analyzer.

Techniques: Activity Assay, Residue, Binding Assay, Variant Assay, Thermal Shift Assay, Fluorescence, Titration, Membrane, SDS Page, Staining, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling

doi: 10.1186/s12964-024-01874-6

Figure Lengend Snippet: Inhibition of BCR::ABL1 signaling in CML cells after AS25 translocation. a Luminescence signal of YopE 1-138 -Monobody-FLAG-HiBiT variants translocated into LgBiT-expressing K562 cells . At time point zero, K562 cells were infected with indicated strains and incubated with NanoLuc substrate Furimazine. Luminescence was followed in 3 min intervals over 2 h. Error area represent mean ± SD of three independent measurements ( n = 3). b Degradation kinetics of YopE 1-138 -Monobody-FLAG-HiBiT variants in LgBiT-expressing K562 cells after delivery. K562 cells were infected with indicated strains and incubated for 2 h. After gentamicin treatment, the long lasting NanoLuc substrate endurazine was added. Luminescence signal was followed in 3 min intervals over 24 h. Error area represents mean ± SD of three independent measurements ( n = 3 ). c Immunoblot analysis to determine intracellular monobody concentrations in K562 cells after infection with the indicated strains. Cell lysates and serial dilutions of recombinant monobody (without secretion signal) were analyzed. Intracellular monobody concentration was calculated based on cell number and cell volume from three independent experiments ( n = 3) and is indicated as mean ± SD. d , e Flow cytometric analysis of STAT5 phosphorylation (pY694) in K562 cells 5 h (d) and 24 h (e) after infection with indicated strains or treatment with BCR::ABL1 inhibitor imatinib. Left panel: Signal intensities (MFI) of cells stained with anti-phospho-STAT5 antibody. Right panel: Quantification of pSTAT5 levels (relative MFI), normalized to untreated, from three independent experiments ( n = 3) plotted as mean ± SD. Ordinary one-way ANOVA followed by Šidák multiple comparisons tests was performed by comparing against the untreated sample. Additional comparison was made between AS25 A57G and AS25 Y45A-A57G . P values below 0.05 were considered statistically significant and asterisks represent statistical significance ( * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001). Only significant results are denoted

Article Snippet: SDS-PAGE gels were blotted on a AmershamTM Protran® Western Blotting nitrocellulose membrane (0.2 μm) [Cytiva (10600001)] using a Trans-Blot Turbo Transfer System [Bio-Rad (1704150)] with the settings: 1.3 A, 25 V, 7 min. Immunoblots were carried out using primary rabbit antibodies against the FLAG peptide (Rockland (600–401-383), 1:5000) in combination with a secondary goat anti-rabbit antibody conjugated to a peroxidase (Sigma (A8275) 1:10,000) and visualized with Immobilon Forte Western HRP substrate (Merck (WBLUF0500)) on a LAS-4000 Luminescence Image Analyzer.

Techniques: Inhibition, Translocation Assay, Expressing, Infection, Incubation, Western Blot, Recombinant, Concentration Assay, Phospho-proteomics, Staining, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling

doi: 10.1186/s12964-024-01874-6

Figure Lengend Snippet: Intracellular target engagement of translocated AS25 monobodies. a Schematic representation of a live cell protein–protein interaction assay with a split-NanoLuc system. Monobodies with secretion signal and SmBiT-peptide are expressed in Y. enterocolitica . The large domain of the Nano-Luciferase (LgBiT) fused to the target protein is stably expressed in the cytosol of eukaryotic cells. Upon infection, the translocation of monobody-SmBiT and interaction of the monobody with its target brings the SmBiT-peptide and the LgBiT domain in close proximity. This leads to complementation and reconstitution of a functional Nano-Luc enzyme, which can be read out by measuring luminescence. b In vitro secretion assay ( n = 3) showing export of YopE 1-138 -AS25-FLAG-HiBiT monobody variants and indicated native T3SS substrates by Y. enterocolitica . Proteins secreted over 180 min were precipitated and analyzed by SDS-PAGE. The secretion deficient strain Δ sctQ was used as control. Left, Coomassie staining of all exported proteins; right, Western blot anti-FLAG. Molecular weight indicated in kDa, expected size of YopE 1-138 -AS25-FLAG-HiBiT (Mb): 28.7 kDa (marked with *). c Expression levels of YopE 1-138 -AS25-FLAG-HiBiT in the indicated strains used in panel b. Western blot anti-FLAG for cellular proteins. d Luminescence measurement of HeLa cells expressing either Abl-SH2-LgBiT (left) or Lck-SH2-LgBiT (right) after infection with the indicated bacterial strains secreting the indicated monobody variants and gentamicin treatment. Results from three independent experiments ( n = 3) performed in triplicates are shown and presented as mean ± SD. Ordinary one-way ANOVA followed with Šidák multiple comparisons tests was performed for the Abl-LgBiT samples against the untreated sample. Additional comparisons were made between AS25 and AS25 A57G and AS25 A57G and AS25 Y45A-A57G . P values below 0.05 were considered statistically significant and asterisks represent statistical significance ( * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001). Only significant results are denoted

Article Snippet: SDS-PAGE gels were blotted on a AmershamTM Protran® Western Blotting nitrocellulose membrane (0.2 μm) [Cytiva (10600001)] using a Trans-Blot Turbo Transfer System [Bio-Rad (1704150)] with the settings: 1.3 A, 25 V, 7 min. Immunoblots were carried out using primary rabbit antibodies against the FLAG peptide (Rockland (600–401-383), 1:5000) in combination with a secondary goat anti-rabbit antibody conjugated to a peroxidase (Sigma (A8275) 1:10,000) and visualized with Immobilon Forte Western HRP substrate (Merck (WBLUF0500)) on a LAS-4000 Luminescence Image Analyzer.

Techniques: Drug discovery, Protein Protein Interaction Assay, Luciferase, Stable Transfection, Infection, Translocation Assay, Functional Assay, In Vitro, SDS Page, Control, Staining, Western Blot, Molecular Weight, Expressing

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