Cy3 conjugated goat anti mouse igg

For immunocytochemistry, coverslips with primary neurons were fixed with 4% paraformaldehyde/4% sucrose for 8 min, washed with PBS, and permeabilized with 0.3% Triton-X100/PBS for 10 min. After blocking in 5% NGS/PBS for 30 min, incubation with primary antibodies followed overnight at 4°C: rb-anti-pan-Synapsin (1:500, E028, T. Südhof, Stanford University), ms-anti-PSD-95 (1:500, NeuroMab), ch-anti-MAP2 (1:10,000, Abcam), rb-anti-Cofilin1 (1:7,000, Abcam), rb-anti-protein-interacting-with-C-kinase-1 (PICK1) (1:500, Proteintech), as well as Alexa568-phalloidin (1:100, Invitrogen), all diluted in 5% NGS/PBS. After washing, cells were incubated with the following secondary antibodies: Alexa488 goat-anti-rabbit IgG, Alexa647 goat-anti-chicken IgG (Invitrogen), Cy3-conjugated goat-anti-mouse IgG (Jackson Immuno Research), diluted 1:500 in 5% NGS/PBS for 1 h at RT. After additional washings in PBS, coverslips were embedded in mounting medium (Dako).

For BrdU incorporation assay, cells were labeled with 10 μm BrdU (Roche, Indianapolis, IN, USA) for 2 h, fixed with 4% para-formaldehyde, and immunostained with anti-BrdU antibody (Roche) followed by staining with Cy™3-conjugated goat anti-mouse IgG (115-165-146; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and counter-stained with DAPI. BrdU-positive cells were scored under a fluorescent microscope and presented as the percentage of BrdU-positive nuclei over total number of nuclei counted. At least 300 nuclei were counted. For immunofluorescence, cells were fixed with 4% paraformaldehyde, immunostained with primary and secondary antibodies in 4% BSA, and counter-stained with DAPI. Antibodies used include anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse Alexa Fluor 488 (A11001; Santa Cruz Biotechnology). Cell images were recorded with an Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with axiovision 3.1 software (Carl Zeiss).

Dulbecco's modified Eagle's medium (DMEM), goat and fetal calf sera (FCS), phosphate-buffered saline (PBS), Paraformaldehyde (PFA), gentamicin, 4',6-diamidino-2-phenylindole (DAPI), and BODIPY 493/503 (Life Technologies, France), Oleic acid (Sigma, France), Mowiol (Calbiochem, France). Mouse monoclonal anti-core antibody ACAP27 (BioRad, France), Cy3-conjugated goat anti-mouse IgG, and HRP-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA), Mouse monoclonal anti-tubulin antibody (Sigma, France) were obtained. Core coding sequence was amplified by PCR from the plasmid pJHF1-CSN6A4 (12 (link)) and subcloned into the expression vector pCI-neo (Promega, France) between a Kozak consensus sequence and a stop codon. Core protein PP mutant expression vector was produced by replacing the codons of residues P138 and P143 with alanine codons by overlapping PCR. Constructs were confirmed by DNA sequencing.

Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Whatman). Membranes were blocked with membrane blocking buffer (MBB: 5% non-fat milk (LabScientific) in PBS containing 0.1% Tween-20) for 1 hour at RT. For chemiluminescent immunodetection of proteins, membranes were first incubated with rabbit anti-Kat60 (0.1–1 μg/mL) or mouse anti-FLAG (2 μg/mL; clone M2; Sigma-Aldrich) antibodies in MBB for 24 hours at 4°C and then incubated with HRP-conjugated goat anti-rabbit IgG (0.1–1 μg/mL; Sigma-Aldrich) or HRP-conjugated goat anti-mouse IgG (2 μg/mL; Sigma-Aldrich) antibodies in MBB for 1 hour at RT. Protein bands were visualized by incubating the membranes with enhanced chemiluminescent substrate (Pierce) and exposing the membranes to autoradiography film (GeneMate). For fluorescent immunodetection of proteins, membranes were first incubated with mouse anti-actin (1:10,000; clone C4; Millipore) or mouse anti-GFP (0.1 μg/mL; clone JL-8; Clontech Laboratories, Inc.) antibodies in MBB for 24 hours at 4°C and then incubated with Cy3-conjugated goat anti-mouse IgG (0.02 μg/mL; Jackson ImmunoResearch Laboratories, Inc.) antibodies in MBB for 1 hour at RT. Protein bands were visualized by fluorescence scanning of the membranes using a variable mode imager (Typhoon Trio; GE Healthcare).