Mouse monoclonal anti vinculin

Cell pellets were suspended in RIPA buffer (5 mM EDTA, 0.15 mol NaCl, 0.1 mol Tris pH 8.0, 1% Triton) with protease and phosphatase inhibitors cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and sonicated three times (for 10 s and 70% amplitude). The samples were incubated on ice for 30 min, and then centrifuged at 10,000× g for 20 min to remove pellet residues. Equal amounts of protein (30 μg/lane) estimated by Bradford protein assay were solubilized in a 2X Laemmli sample buffer containing 20% of 2-mercaptoethanol. Proteins were separated by SDS-PAGE on 10–12% polyacrylamide gel and then electro-transferred to nitrocellulose membranes (Trans-Blot Turbo Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Membranes were, then, incubated overnight at 4 °C with rabbit polyclonal anti-α-actin (Abcam, Cambridge, UK) and with mouse monoclonal anti-vinculin (Santa-Cruz Biotechnologies, Dallas, TX, USA). Thus, membranes were incubated for 1 h with HRP-conjugated secondary antibody (1:3000; Bio-Rad, CA, USA). The immune complexes were detected by enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis of the bands was performed using Image J software. The results were expressed as arbitrary unit (A.U.) and represented as mean of three independent experiments.

The following antibodies were used for immunofluorescence: mouse monoclonal anti-human/mouse proinsulin biotinylated antibody (1:200; R&D Systems (Minneapolis, MN, USA), #BAM13361), mouse monoclonal anti-calnexin antibody (1:100; Novus Biologicals (Englewood, CO, USA), #NB300-518), rabbit polyclonal anti-GM130 antibody (1:200; Novus Biologicals, #NBP2-53420), guinea pig polyclonal anti-insulin antibody (1:50; GeneTex (Zeeland, MI, USA), #GTX27842). Secondary antibodies were from ThermoFisher Scientific: anti-streptavidin AlexaFluor-568 (1:200; #S11226), AlexaFluor-488 (1:200; #A11001 and #A11073) and AlexaFluor-568 (1:200; #A10042).
The antibodies used for immunoblotting were the following: mouse monoclonal anti-actin antibody (C4) (1:500), rat monoclonal anti-GRP78/Bip (1:250) and mouse monoclonal anti-vinculin (1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA), rabbit polyclonal anti-Phospho-eIF2α (1:500), rabbit polyclonal anti-eIF2α (1:500) and mouse monoclonal anti-Chop (clone L63F7) (1:200) from Cell Signaling (Danvers, MA, USA), rabbit polyclonal anti-Atf4 (1:1000) from GeneTex. Secondary antibodies conjugated with HRP were from Biolegend (anti-mouse and anti-rabbit) and from Santa Cruz Biotechnology (anti-rat). Standard blocking conditions (5% milk in TBS-T) were used throughout, except when anti-P-eIF2α antibody was used, and 1% BSA in TBS-T was utilized.

Cell lysis and Western blot analysis were performed as described previously [40 (link)], using the following antibodies: Rabbit polyclonal anti-SIK2 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-SIK2-pS385 (Kinexus, Sydney, Australia), rabbit polyclonal anti-AKT (Cell Signaling), rabbit polyclonal anti-AKT-pS473 (Cell Signaling), mouse monoclonal anti-β-actin (Sigma-Aldrich), rabbit polyclonal-anti KIF18B (ThermoFisher scientific, Waltham, MA, USA), rabbit polyclonal anti-EB1 (Abcam, Cambridge, UK), mouse monoclonal anti-cyclin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-Histone 3-pS10 (ThermoFisher scientific), mouse monoclonal anti-PLK1 (Santa Cruz Biotechnology), mouse monoclonal anti-vinculin (Santa Cruz Biotech), mouse monoclonal anti-P53 (Santa Cruz Biotech), rabbit polyclonal anti-P21 (cell signaling), rabbit polyclonal anti-BCL-xL (Santa Cruz Biotechnology), rabbit polyclonal anti-Cyclin D (Santa Cruz Biotechnology).
Reagents were purchased from the following sources: Paclitaxel (T7402) Sigma-Aldrich, propidium iodide (440300250) Acros Organics (Fair Lawn, NJ, USA), RNase A (1007885) Qiagen (Hilden, Germany), PE Annexin V (556421) and 7AAD (21-68981E) BD Biosciences, Caspase-Glo^® 3/7 Assay system (P1781) Promega, RO3306 (S7747) Selleckchem (Houston, TX, USA).