Monoclonal igg mouse anti vinculin

For the estimation of GBM invasion/cytoskeleton-related vinculin rearrangements, the cells subjected to 72 h incubation with EGF/erlotinib were harvested with cold (~4 °C) Ca²⁺/Mg²⁺-free PBS/EDTA solution, centrifuged, and dissolved in cell lysis buffer with protease inhibitor cocktail, followed by the freeze–thaw procedure and sonication. A Bradford assay was used for determination of the total protein content in the obtained lysates. Samples (20 µg of protein) were separated on 12% polyacrylamide gel (SDS-PAGE electrophoresis; Laemmli protocol [31 (link)]), followed by electrotransfer onto PVDF membranes (Immun-Blot^® PVDF Membrane, #1620177; Bio-Rad, Hercules, CA). Membranes were blocked with skimmed milk/TBST solution. For immunodetection, the monoclonal IgG mouse anti-vinculin (Sigma; No. V9131; 1:500) was used. α-tubulin, labeled with monoclonal IgG mouse anti-α-tubulin antibody (Sigma; No. T9026; 1:1000) was used as a reference protein. Signal detection was performed with HRP-conjugated antibodies: HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific; No. 31430), followed by chemiluminescent HRP substrate incubation (Merck, Luminata Crescendo; No. WBLUR0500). For membrane imaging, a MicroChemi system (SNR Bio-Imaging System) was used.

The cells were cultured in 12 well plates on UVC-sterilized coverslips and treated with the relevant concentrations of EGF/Erl/NAC for 72 h. For immunolocalization, the cells were fixed with 3.7% formaldehyde, followed by 0.1% Triton X-100 permeabilization. Blocking of non-specific binding sites was performed with 2% BSA incubation for 30–45 min in 37 °C. Specimens were incubated for 45 min with monoclonal IgG mouse anti-vinculin (Sigma; No. V9131; 1:300) antibody diluted in 2% BSA and 0.01% Tween. After washing (2% BSA), the sets of secondary antibodies/specific dyes were applied for 45 min: AlexaFluor488-conjugated donkey anti-mouse IgG (Invitrogen; No. A21202), AlexaFluor546-conjugated phalloidin (Invitrogen, No. A22283) for F-actin visualization, and Hoechst 33258 (Sigma) for DNA staining. Images were acquired with a Leica DMI6000B fluorescence microscope equipped with a DFC360FX CCD camera and total internal reflection fluorescence (TIRF) module. Z-stack scanning followed by 2D deconvolution (LAS X; Leica) were performed for high-resolution vinculin/cortical F-actin co-localization. Raw images were additionally processed (contrast adjustment, background subtraction, fluorimetric analysis, circularity calculation) in ImageJ software.