Mouse anti clathrin heavy chain

The antibodies and reagents used in this study included the following: Alexa Fluor 594- and 488-conjugated phalloidin (Invitrogen); CellTracker Blue (Invitrogen); BODIPY-lactosylceramide (Invitrogen); Alexa Fluor 594- and 488-conjugated goat anti-rabbit and goat anti-mouse antibodies (Invitrogen) (2 μg/ml); rabbit anti-caveolin-1 (Abcam, ab2910) (10 μg/ml for immunofluorescence); mouse anti-clathrin heavy chain (BD Biosciences, 610499) (5 μg/ml for immunofluorescence); mouse anti-α-tubulin (Developmental Studies Hybridoma Bank [DSHB], 12G10) (1:1,000 for Western blotting); mouse anti-ezrin (Developmental Studies Hybridoma Bank, CPTC-Ezrin-1) (1:100 for immunofluorescence); rabbit anti-Listeria monocytogenes (BD Difco, 223021) (1:300 for immunofluorescence); horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit antibodies (Invitrogen) (1 μg/ml). The mouse monoclonal anti-α-tubulin antibody (12G10) was deposited into the DSHB by J. Frankel and E. M. Nelsen. The mouse monoclonal anti-ezrin antibody (CPTC-Ezrin-1) was deposited into the DSHB by Clinical Proteomics Technologies for Cancer. The mouse anti-epsin-1 antibody (clone zz3) (1:10 dilution for immunofluorescence) was generated previously (86 (link)).

C2C12 myoblasts were seeded on gelatin coated coverslips and differentiated as previously described. Lysotracker 75 nM was added for 45 min when specified. After two washes with PBS, cells were fixed with 4% paraformaldehyde for 10 min. After three PBS washes, cells were permeabilized with 0.1% Triton X-100 and washed thrice with PBS. Coverslips were incubated with blocking solution (5% Fetal Bovine Serum, 1% Albumine from Bovine Serum and 0.02% sodium azide) overnight at 4°C. Subsequently, cells were incubated for 2 h at RT in a hydration chamber with the following primary antibodies: mouse anti-Aβ 6E10 1:200, rabbit anti-oligomers A11 1:200 (Invitrogen), mouse anti-phospho-Akt Ser473 1:100, rabbit anti-Akt 1:100 and mouse anti-clathrin heavy chain 1:200 (BD Biosciences). Then, cells were washed thrice with blocking buffer and incubated with Alexa Fluor 488 or 555 goat anti-mouse antibodies (Invitrogen) 1:1000 for 1 h at RT. When specified, cells were incubated with 1μM Topro-3 for 15 min, washed with PBS and mounted with Mowiol. Digital images were taken at RT with a Leica TCS SP confocal microscope with ×40 and ×63 objective and analyzed with Leica confocal software (Heidelberg, Germany) and Image J software.

Rabbit polyclonal anti-ABCG1 and anti-flotillin-1 antibodies were purchased from Santa Cruz (Santa Cruz, CA). Rabbit polyclonal anti-caveolin-1, mouse anti-clathrin heavy chain, and mouse anti-FAK antibodies were obtained from BD Transduction Laboratory (Lexington, KY). Mouse anti-paxillin antibody and a cholera toxin subunit B Alexa 555 conjugate were purchased from Invitrogen (Carlsbad, CA). An anti-ABCA1 antibody was provided by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). HDL was acquired from Calbiochem (San Diego, CA). Human embryonic kidney (HEK) 293 cells and SH-SY5Y cells were obtained from American Type Culture Collection (Manassas, VA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO), GE Healthcare (Little Chalfont, UK), Cayman Chemical (Ann Arbor, MI), Wako Pure Chemical Industries (Osaka, Japan), and Nacalai Tesque (Kyoto, Japan).