Synaptotagmin

E1 (31 (link)), a polyclonal antibody specific to human tau (aa 19-33, unphosphorylated), was prepared in our laboratory. MS06, a polyclonal antibody specific to mouse tau, was raised against mouse tau polypeptide corresponding to amino acid residue 118–131 (SKDRTGNDEKKAKG). Tau5, pS199, pT231, and pS396 were purchased from Biosource International (Camarillo, CA, USA). Tau1 was from Chemicon (Temecula, CA, USA). Monoclonal antibodies to β-actin and β-tubulin were purchased from Sigma. Monoclonal antibodies to GAP-43, PSD-95, and synaptotagmin were purchased from BD Transduction Laboratories (San Jose, CA, USA). For western blotting, antibodies were used at the following dilutions in blocking solution: E1, 1:5,000; MS06, 1:2,000; Tau1, 1:5,000; Tau5, 1:2,000; pS199, 1:5,000; pT231, 1:2,000; pS396, 1:2,000; β-actin, 1:5,000; β-tubulin, 1:5,000; GAP-43, 1:2,000; PSD-95, 1:2,000; synaptotagmin, 1:2,000.

Protease inhibitor cocktail (Sigma, 4693132001) and protein G-Agrose (Roche, 11719416001), and antibodies against KLC (Chemicon, MAB1616), Kif17 (Sigma, K3638), CHC (BD Biosciences, 610500), Cla (Santa Cruz), Clb (Santa Cruz), Hsc70 (Santa Cruz, sc7298 and Enzo, ADI-SPA-816-D), α-Adaptin (BD Biosciences 610502), γ-Adaptin (BD Biosciences 610386), Dynamin I (Santa Cruz, sc-12724), Syntaxin 6 (BD Transduction Laboratories, 610636), Synaptotagmin (BD Transduction Laboratories, 610433), EEA1 (UPSTATE, 07-292), Auxilin (Santa Cruz, sc104213), Hsp40 (Enzo Life Sciences, ADI-SPA-400-D), Beta Actin (Sigma, A5316), and VSV-G (Abcam, ab1874) were used. The antibody against Kif5b was previously described^{63 (link),64 (link)}. The following secondary antibodies for immunofluorescence staining were used: Cy3 donkey anti-mouse (Jackson Immunoresesarch), Cy3 donkey anti-rabbit (Jackson Immunoresesarch), Alexa Fluor 488 donkey anti-mouse (Jackson Immunoresearch) and Alexa Fluor 488 donkey anti-rabbit (Jackson Immunoresearch). Cell-penetrating peptides synthesized in GL biochem (Shanghai) Ltd are: TAT-Kif5b^891–915 peptide GRKKRRQRRRPPQDRKRYQQEVDRIKEAVRSKNMARRG and the control TAT-scramble peptide: GRKKRRQRRRPPQRDVRDRGANKIQAREYRQRKVMESK. The GST-fused Kif5b fragments were generated by PCR amplification of pCDNA3.Kif5b plasmid and subcloned into pGEX-4T-1 (Novagen).