anti flag m2 primary antibody  (Merck & Co)

Journal: Molecular Pharmacology

Article Title: Interaction between W41 of the hepatitis B virus preS1 surface peptide and Y146/F274 of the cellular receptor molecule Na + /taurocholate co-transporting polypeptide is essential for virus entry

doi: 10.1016/j.molpha.2025.100069

Figure Lengend Snippet: Plasma membrane expression of the NTCP-Y146A/F274A mutant. (A) Immunofluorescence staining with the anti-FLAG antibody (green fluorescence) localized the NTCP-Y146A/F274A mutant protein to the plasma membrane of HEK293 cells comparable to the localization of the NTCPwt protein. Nuclei were stained with Hoechst 33342 (blue). Images represent maximum projections of z-stacks at 63× magnification after deconvolution. Scale bar, 10 μ m. (B) Western blot expression analysis of the NTCPwt and NTCP-Y146A/F274A mutant proteins. Proteins were extracted from the respective NTCPwt-HEK293, NTCP-Y146A/F274A-HEK293, or nontransfected HEK293 cells with the ProteoExtract membrane protein extraction kit and were subjected to enzymatic deglycosylation with PNGase before polyacrylamide gel separation. Expression of GAPDH served as loading control. No NTCP signals were detected in the cytoplasmic fraction.

Article Snippet: Immunofluorescence staining of NTCP expression of the transfected HepG2 cells was done with mouse monoclonal anti-FLAG M2 primary antibody (Merck, RRID AB_262044 ) and a goat anti-mouse IgG (H+L) secondary antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, RRID AB_2534057 ), along with 4′,6-diamidino-2-phenylindole staining to determine the number of nuclei for further comparison of transfection efficiency.

Techniques: Clinical Proteomics, Membrane, Expressing, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Western Blot, Protein Extraction, Control

Journal: Molecular Pharmacology

Article Title: Interaction between W41 of the hepatitis B virus preS1 surface peptide and Y146/F274 of the cellular receptor molecule Na + /taurocholate co-transporting polypeptide is essential for virus entry

doi: 10.1016/j.molpha.2025.100069

Figure Lengend Snippet: In vitro HBV infection of NTCP-transfected HepG2 cells. HepG2 cells were transiently transfected with either NTCPwt or the Y146A/F274A-NTCP mutant. This transfection was performed in 3 independent experiments, which varied in their absolute transfection rates. Therefore, representative data from 1 out of 3 independent experiments are shown, rather than combined data from all experiments. (A) To assess functional NTCP expression after transfection, transport studies were performed with the fluorescent bile acid 3 β -NBD-TCA and anti-FLAG immunofluorescence was used to directly detect the expressed wild-type and mutant NTCP proteins. For both assays, the number of fluorescent cells was quantified by automated image analysis on 6 different fluorescent images. Data represent means ± SD and statistical significance was analyzed by Student’s t test with P < .05. (B) In vitro HBV infection of NTCPwt- and Y146A/F274A-NTCP-transfected HepG2 cells and inhibition with the wild-type (genotype D) myr-preS1 peptide or its W41G mutant at increasing concentrations. Before HBV infection, NTCP-transfected HepG2 cells were preincubated with 10, 100, or 500 nM of the respective myr-preS1 peptides for 1 hour. Subsequently, cells were infected with cell-culture-produced HBV (subgenotype D3) derived from a stably transfected inducible HepG2 cell line as reported. Cells were incubated with the virus inoculum and the respective myr-preS1 peptide for 24 hours. After infection, the cell culture medium was renewed every 2 to 3 days. Using the supernatants collected on day 10 post infection, HBeAg secreted by infected cells was quantified. Data represent means ± SD of triplicate determinations. ∗Significant difference between the NTCPwt- and the NTCP-Y146A/F274A-expressing HepG2 cells and # significant inhibition in the NTCPwt-HepG2 cells compared with control (without preS1) with P < .05 according to two-way ANOVA.

Article Snippet: Immunofluorescence staining of NTCP expression of the transfected HepG2 cells was done with mouse monoclonal anti-FLAG M2 primary antibody (Merck, RRID AB_262044 ) and a goat anti-mouse IgG (H+L) secondary antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, RRID AB_2534057 ), along with 4′,6-diamidino-2-phenylindole staining to determine the number of nuclei for further comparison of transfection efficiency.

Techniques: In Vitro, Infection, Transfection, Mutagenesis, Functional Assay, Expressing, Immunofluorescence, Inhibition, Cell Culture, Produced, Derivative Assay, Stable Transfection, Incubation, Virus, Control