Figure 2.
p600 is a novel MAP. A, Amounts of p600 in the cytosolic fraction (S2), pellet 1 (P1), or synaptic compartment (P2). H, Homogenate; S1, preclear lysate. The amount of each protein in each specific fraction is estimated in percentage. B, p600 is a soluble protein and cofractionates with membranes. The ER marker Bip is used as control. C, p600 is not found in preparations of NFs from adult spinal cord and brain. D, p600 is found in the active MT fraction (S) and is enriched in the fraction P2 containing tubulin and MAPs. I, Input; P1, fraction containing insoluble aggregates and debris with no tubulin. E, Immunogold labeling of MTs from cytosol-depleted primary mouse cortical neurons stabilized with Taxol using α-tubulin or p600 antibodies. Scale bar, 50 nm. F, Western blots depicting the expression of the seven FLAG-tagged fragments of p600 (designed A to G) in the neuroblastoma CAD cell line as detected with FLAG antibody (red stars). G, MT preparations from CAD cells transfected with each of one of the seven fragments of p600. Fragments E and G were found in significant amounts in MT preparations (red stars). Tubulin and actin, Loading controls; Tau, positive control; black stars refer to nonspecific bands. H, Bacterially expressed and purified fragments E, F, and G, but not B or D, enhance MT polymerization in vitro as detected by absorbance at 340 nm. Four to six independent assays were performed for each fragment. A representative assay is depicted for each fragment. I, p600 RNAi nos. 1 and 2 efficiently knockdown p600 protein. Knockdown of p600 in CAD cells by RNAi no. 1 or 2 reduces the levels of acetylated tubulin (Ac-Tub), but not tubulin or other cytoskeletal proteins. Five samples for each condition were processed for quantifications. Error bars indicate SDs.