Mouse anti vinculin

Immunoblotting was carried out in different cellular fractions (total cell lysate, isolated focal adhesion fractions and cell body fractions) after determination of protein concentrations using the DC™ Protein Assay (Bio-Rad, 5000116) 31 (link). Briefly, the cell lysates containing equivalent amounts of protein were loaded and separated by SDS-PAGE and immunodetected with antibodies: rabbit anti-GPR124 (TEM5, 1:1,500, ThermoFisher, PA5-20442), mouse anti-Vinculin (1:3,000, Millipore, MAB3574), mouse anti-Flag (1:500, Sigma-Aldrich, F3165), mouse anti- Paxillin (1:3,000, ThermoFisher, MA5-13356), mouse anti-β-actin (1:5,000, Cell Signaling Technology, 3700). After incubation for 12 h at 4 °C, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactivity was visualized by enhanced chemiluminescence (Amersham Life Science).

Hand-picked islets were washed once with PBS and then resuspended in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors cocktails (Sigma) as described^{32 (link)}. Islets were lysed by repeated pipetting or triturating through a 30G syringe needle. Lysates were resolved on a 4–12% NuPAGE Bis-Tris gel, and then electrotransferred to nitrocellulose membrane for western blotting. Antibodies used were mouse anti-proinsulin (ALPCO), guinea pig anti-insulin (Covance), and mouse anti-vinculin (Millipore). Horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc., with protein visualized by ECL (Millipore).

Protein extraction and Western blot analysis of whole mouse cerebellar and cortical tissue was performed as previously described [29 (link)]. The following primary antibodies were used: mouse anti-Gapdh (Sigma G8795, 1:10,000, St. Louis, MO, USA), mouse anti-Vinculin (Millipore v9264 1:10,000, Burlington, MA, USA), rabbit anti-Gfap (Sigma G4546, 1:500), rabbit anti-Iba1 (Wako, 019-19741, 1:400), rabbit anti-Prkcb (Thermo Fisher Scientific 12919-1-AP, 1:1000, Waltham, MA, USA), rabbit anti-Camkk2 (Thermo Fisher Scientific 11549-1-AP, 1:1000).

Gelatin coverslips were prepared as published previously [33 (link)]. Briefly coverslips were with treated with 50 μg/ml poly-L-lysine, then 0.5% glutaraldehyde, then 0.2% Oregon green gelatin (Molecular Probes, Eugene, OR, USA), then 5 mg/ml NaBH₄, with PBS washes done between each treatment. Rheumatoid synovial fibroblasts (7.5 × 10³) were plated on the coverslips in media with 10% FBS; incubated for 2, 5 or 24 hours; fixed with 3% formaldehyde; quenched with 0.15 M glycine; permeabilized with 0.2% Triton-X 100; blocked with 5% goat serum; and incubated with mouse anti-cortactin (1:200, clone 4F11; EMD Millipore, Billerica, MA, USA), mouse anti-vinculin (1:500, clone V284; EMD Millipore) and rabbit anti-paxillin pY118 (1:200, polyclonal; BioSource International, Camarillo, CA, USA), followed by washing and incubating with either goat anti-mouse rhodamine red or donkey anti-rabbit tetramethylrhodamine (1:250). Coverslips were mounted and viewed at 400× magnification with an inverted fluorescence microscope (Nikon Eclipse TE300; Nikon Instruments, Melville, NY, USA). Images were digitally acquired and processed with MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Standard methodologies were used. Protein extracts were separated by 10% or 8% SDS-PAGE and transferred to a PVDF membrane. Membranes were incubated using the following specific antibodies, including mouse anti-puromycin (1:500, DSHB), mouse anti-Vinculin (1:2000, Merck), mouse anti-GAPDH (1:2000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-APP (1:2000, Merck), rabbit anti-ADAM10 (1:500, Abcam, Cambridge, UK), mouse anti-sAPPα (1:500, IBL America, Minneapolis, MN, USA), rabbit anti-OCT3/4 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-MAP2 (1:2000, Merck), mouse anti-Nestin (1:1000 Santa Cruz Biotechnology), mouse anti-SAP97 (1:1000, ENZO Life Sciences, Farmingdale, NY, USA) and rabbit anti-FMRP (1:1000, produced in house PZ1 [52 (link)]), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:5000, Cell Signaling Technology, Danvers, MA, USA). Proteins were revealed using an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) and the imaging system LAS-4000 mini (GE Healthcare, Chicago, IL, USA). Quantification was performed using the IQ ImageQuant TL software (GE Healthcare). Detection of GAPDH, Vinculin, and Coomassie staining were used as normalizers. For all SDS-PAGE PageRuler™ Plus Prestained Protein Ladder (10 to 250 kDa, Thermo Fisher Scientific) was used.