P1663 antibody

N2a cells were washed with PBS and lysed in a modified radioimmunoprecipitation assay (RIPA) lysis buffer (#9806, Cell Signaling Technology) with 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentrations were determined with a BCA Protein Assay kit (#23227, Thermo). An equal amount of protein lysate was separated by 8% or 10% SDS-polyacrylamide gels and transferred to PVDF membranes (3,010,040,001, Roche). Membranes were blocked in TBST containing 5% non-fat dried milk and incubated with primary antibodies overnight at 4 °C. The membranes were washed with TBST and incubated with secondary antibody (1:2000 dilutions in 5% non-fat dried milk) for 2 h at room temperature (RT). Bound antibodies were visualized by chemiluminescent HRP substrate (#32209, Thermo). The mean densities of protein bands were measured by Image J software. The primary antibodies used are as follows: anti-rabies Virus (5B12) (NB110–7542, Novus) (1:1000), GAPDH (1A6) mAb (MB001, Bioworld) (1:5000), Clathrin Heavy Chain (P1663) Antibody (#2410, Cell Signaling Technology) (1:500), Caveolin-1 Antibody (#3238, Cell Signaling Technology) (1:500).

After fixation the coverslips were washed in stabilization buffer once and fixation was quenched with 50 mM NH4Cl in stabilization buffer for 7 min and washed two more times. Cells were blocked for 1 h with 4% (v/v) horse serum and 1% (w/v) bovine serum albumin (BSA) in stabilization buffer. The samples were then incubated with anti-clathrin heavy chain (P1663) antibody (1:100 dilution, #2410, Cell Signaling Technology) and 1% BSA in stabilization buffer at 21 °C for 1 h followed by 4 washing steps with 1% BSA in stabilization buffer. Next, cells were incubated with goat anti-rabbit IgG Alexa Fluor 568 (1:500 dilution, #A-11011, Invitrogen) and CellMask Deep Red Plasma Membrane Stain (1:5000, # C10046, Invitrogen) with 1% BSA in stabilization buffer for 45 min. Samples were rinsed 4 times with stabilization buffer, postfixed in 4% (w/v) paraformaldehyde in stabilization buffer for 10 min and quenched as described above prior to imaging by spinning disc confocal microscopy.