Journal: International Journal of Dentistry

Article Title: Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland

doi: 10.1155/2023/1765317

Figure Lengend Snippet: Mouse parotid acinar cells in primary culture. (a) Cell proliferative capacity of BMP2-added (100 ng/mL) and nonadded (control) groups after 48 hr of culture. The proliferative capacity of BMP2-added cultured cells was significantly increased compared to that in the control group. ∗∗ Indicates significance at P < 0.01, ∗ P < 0.05. Data were shown as mean ± SD. Three independent experiments were performed. (b) Protein expression of E-cadherin (an epithelial marker) and vimentin (a mesenchymal marker) in cultured cells of each group at 48 hr after the addition of BMP2. NIH3T3 (3T3) was used as a positive control for mesenchymal markers. RS: rat serum.

Article Snippet: NIH3T3 whole cell lysate (Novus Biologicals, Centennial, CO, USA) was used as a positive control for mesenchymal markers.

Techniques: Control, Cell Culture, Expressing, Marker, Positive Control

Journal: Cell calcium

Article Title: Ca 2+ -dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

doi: 10.1016/j.ceca.2021.102457

Figure Lengend Snippet: Fig. 1. Interaction of S100A6 with cofilin-1 in NIH3T3 fibroblasts. (A) Pull-down assay with the use of protein lysate from NIH3T3 cells and S100A6 affinity resin (upper panel) or empty resin (lower panel). Lanes: 1-input, 2-unbound fraction, 3-last wash, 4-first wash with 0.5 M NaCl, 5-last wash with 0.5 M NaCl, 6-first wash with 1 M NaCl, 7- last wash with 1 M NaCl, 8- elution in buffer containing EGTA. Fractions were analyzed by SDS-PAGE (15% gel) fol lowed by immunoblotting developed with anti- cofilin-1 antibody. (B) Co-immunoprecipitation of S100A6 with cofilin-1 from NIH3T3 cell lysate. 30 μg of protein lysate was used directly for immunoblotting (input; lane 1 in both upper and lower panel) and 2.5 mg of protein lysate was incubated with (upper panel) or without (control, lower panel) anti-S100A6 monoclonal antibody and then with protein A/G agarose. In both panels, lane 2 shows unbound fraction, lane 3-last wash and lane 4-elution. Proteins were identified by immunoblotting using anti- cofilin-1 antibody. (C) Presence of S100A6- cofilin-1 complexes in NIH3T3 cells studied by PLA. Complexes of examined proteins are visualized in red; cell nuclei, stained with DAPI, are in blue. Scale bar is 20 μm.

Article Snippet: For co-immunoprecipitation assays 2.5 mg of protein lysate from NIH3T3 cells, obtained using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer’s instruction was incubated with protein A/G-Agarose (Santa Cruz Biotechnology) for 1 h at 4◦C, as described by Jurewicz et al. [31].

Techniques: Pull Down Assay, SDS Page, Western Blot, Immunoprecipitation, Incubation, Control, Staining

Journal: Infection and Immunity

Article Title: Escherichia coli Cytotoxic Necrotizing Factor and Pasteurella multocida Toxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association

doi: 10.1128/iai.69.9.5931-5935.2001

Figure Lengend Snippet: FIG. 1. Dose- and time-dependent induction of tyrosine 397 autophosphorylation of FAK by CNF1 and PMT. (A) Quiescent Swiss 3T3 cells were treated with purified PMT over a concentration range of 0.01 to 5 ng ml21, with lysates from recombinant E. coli expressing CNF1 over a concentration range of 0.01 to 5 mg ml21, or with lysates from E. coli harboring pBluescript (pBS) for 4 h. The cellular FAK was immunopre- cipitated, and the isolated proteins were separated by SDS-PAGE. A Western blot of the gel was probed with a polyclonal antiserum specific for the Tyr397 autophosphorylation site (FAKPY397). The antibodies were stripped from the membranes and probed with the anti-FAK antibody (FAK). (B) Quiescent Swiss 3T3 cells were treated with lysates from E. coli expressing CNF1 or harboring control pBluescript plasmid at a final concentration of 0.5 mg ml21 or with PMT or the C1165S mutant PMT (PMTc-s) at a final concentration of 5 ng ml21. Cells were harvested before the toxin preparations were added and at 1, 2, 3, 4, 6, and 8 h after addition of the toxins. The cellular FAK was immunoprecipitated, and the isolated proteins were separated by SDS-PAGE. A Western blot of the gel was probed with a polyclonal antiserum specific for the Tyr397

Article Snippet: The stimulation of FAK phosphorylation at Tyr by PMT or CNF1 was investigated, as described previously (41), by exposing quiescent Swiss 3T3 cells to each of the toxins at different concentrations for 4 h. After treatment, the cells were solubilized and FAK was immunoprecipitated from the cleared lysate by using polyclonal rabbit anti-FAK antibody C-20 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Recombinant, Expressing, Isolation, SDS Page, Western Blot, Control, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: Infection and Immunity

Article Title: Escherichia coli Cytotoxic Necrotizing Factor and Pasteurella multocida Toxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association

doi: 10.1128/iai.69.9.5931-5935.2001

Figure Lengend Snippet: FIG. 2. Induction of FAK-Src association by CNF1 and PMT. (A) Quiescent Swiss 3T3 cells were either left untreated (Unt) or were treated with PMT or the C1165S mutant (PMTc-s) at a final concen- tration of 5 ng ml21 or with lysates from E. coli expressing CNF1 or harboring the control pBluescript plasmid (pBS) at a final concentra- tion of 1 mg ml21. Cells were harvested after 4 h. The cellular Src was immunoprecipitated, and the isolated proteins were separated by SDS- PAGE. A Western blot of the gel was probed with a polyclonal anti- FAK antibody (FAK) to detect FAK that had coimmunoprecipitated with Src. (B) Quiescent Swiss 3T3 cells were treated with bombesin at a final concentration of 10 nM or with lysates from E. coli expressing CNF1 at a final concentration of 1 mg ml21 for 15 min or 4 h, respec- tively, or were left untreated for 4 h. FAK coimmunoprecipitating with Src was detected as described above for panel A.

Article Snippet: The stimulation of FAK phosphorylation at Tyr by PMT or CNF1 was investigated, as described previously (41), by exposing quiescent Swiss 3T3 cells to each of the toxins at different concentrations for 4 h. After treatment, the cells were solubilized and FAK was immunoprecipitated from the cleared lysate by using polyclonal rabbit anti-FAK antibody C-20 (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing, Control, Plasmid Preparation, Immunoprecipitation, Isolation, SDS Page, Western Blot, Concentration Assay

Journal: Infection and Immunity

Article Title: Escherichia coli Cytotoxic Necrotizing Factor and Pasteurella multocida Toxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association

doi: 10.1128/iai.69.9.5931-5935.2001

Figure Lengend Snippet: FIG. 3. Effect of p160/ROCK inhibitors on CNF1- and PMT-induced FAK autophosphorylation and actin stress fiber formation. (A) Quiescent Swiss 3T3 cells were incubated in DMEM with p160/ROCK inhibitors HA1077 (2.0 and 10 mM) and Y-27632 (0.5, 2.0, and 10 mM) for 1 h prior to the addition of a lysate from E. coli expressing CNF1 at a final concentration of 0.5 mg ml21. Cells were harvested after 4 h. The cellular FAK was immunoprecipitated, and the isolated proteins were separated by SDS-PAGE. A Western blot of the gel was probed with a polyclonal antiserum specific for the Tyr397 autophosphorylation site (FAKPY397). The antibodies were stripped from the membranes and reprobed with the anti-FAK antibody (FAK). Cells were similarly pretreated (1) or not pretreated (2) for 4 h with Y-27632 (B) or HA1077 (C) at a final concentration of 10 mM prior to the addition of wild-type PMT or mutant C1165S PMT (PMTc-s) at a final concentration of 5 ng ml21. The basal level of Tyr397 phosphorylation was determined from cells not treated with toxin (Unt). (D) Quiescent Swiss 3T3 cells were incubated in DMEM with (1) or without (2) p160/ROCK inhibitor Y-27632 (10 mM) for 1 h prior to the addition of lysate from E. coli harboring control pBluescript plasmid or expressing CNF1 at a final concentration of 0.5 mg ml21 or wild-type or mutant PMT at a final concentration of 5 ng ml21 or not treated with a toxin. After 16 h, the cells were fixed in paraformaldehyde (3.7%), permeabilized, and stained with phalloidin-rhodamine to demonstrate actin organization. The cells were examined by fluorescence microscopy.

Article Snippet: The stimulation of FAK phosphorylation at Tyr by PMT or CNF1 was investigated, as described previously (41), by exposing quiescent Swiss 3T3 cells to each of the toxins at different concentrations for 4 h. After treatment, the cells were solubilized and FAK was immunoprecipitated from the cleared lysate by using polyclonal rabbit anti-FAK antibody C-20 (Santa Cruz Biotechnology).

Techniques: Incubation, Expressing, Concentration Assay, Immunoprecipitation, Isolation, SDS Page, Western Blot, Mutagenesis, Phospho-proteomics, Control, Plasmid Preparation, Staining, Microscopy

Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours. Under these conditions EHD1 was seen to co-localize with SMO in the primary cilia. A profile scan for SMO and EHD1 in the primary cilia merged panel is shown. Under these conditions, EHD1 was seen to traffic to the cilia of 60% cells studied. ( B ) WT NIH3T3 cells stably expressing SMO-GFP and transiently expressing EHD1-DsRed were starved in low serum media for 24 hours and stimulated with SAG immediately before starting live imaging of Smoothened and EHD1.As reported in earlier studies, Smoothened was found in preciliary vesicles under non-stimulated conditions but upon SHH pathway activations, EHD1 was seen to associate with Smoothened vesicles and co-traffic with SMO into the primary cilia.

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Stable Transfection, Expressing, Imaging

Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) Immunoblotting was used to assess the presence of SMO in anti-EHD1 immunoprecipitates from WT NIH3T3 cells stably expressing SMO GFP treated with SAG (24 hrs). Input samples show the amount of each protein in the whole extract before the IP. Immunoblots showing the amount of SMO that co-precipitated with EHD1 from cells that stably expressed the protein and were treated with SAG for 24 hrs. A control IP was performed with non-specific rabbit IgG instead of EHD1 antibody and WT NIH3T3 cell lysates stably expressing SMO GFP treated with SAG (24 hrs). Arrows denote the molecular weight fraction of SMO that associates with EHD1. Full-length blots/gels are presented in . ( B ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours and lysed and these lysates were incubated with GST-EHD1 or GST-EH domain deleted EHD1. The membranes were probed with antibodies to Smoothened and GST.GST-fused to the empty plasmid vector was used as a negative control. The data is representative of three separate experimental repeats. Full-length blots/gels are presented in .

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Western Blot, Stable Transfection, Expressing, Control, Molecular Weight, Incubation, Plasmid Preparation, Negative Control