Extended Data Fig. 3. Microtubule sedimentation assays of ER proteins.

a, Microtubule co-sedimentation assays of U2OS cells. The pellet (P) of 37 °C incubation indicates the microtubule-bound fraction, and supernatant (S) indicates the unbound fraction. 4 °C incubation acts as a microtubule-free control. b, Microtubule co-sedimentation assays of U2OS cells with exogenous CLIMP63-HA, p180s-myc or KTN1-myc expression. Proteins were expressed in corresponding knockout cells. Note that only one representative α-tubulin blot (from the CLIMP63 assay) is shown. c, p180 is very unstable after cell lysis. The input sample was collected by directly adding sample buffer (50 mM Tris, pH 6.8, 1 mM DTT, 10% glycerol, 2% SDS, 0.1% Bromophenol Blue) onto the plate followed by immediate boiling. Other samples were incubated in lysis buffer (50 mM Tris, pH7.4, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail) at room temperature or on ice for the indicated times before adding sample buffer and boiling. d, Western blotting of WT or p180 knockout (KO) U2OS or COS7 cells, showing that only the long isoform is detectable in these cell lines. e, Detailed mapping of microtubule-binding domains of CLIMP63. f, Mapping of microtubule-binding domains of p180. Amino acid sequences around key microtubule-binding sites are shown at the bottom. Note that this part of the sequence is present in both long and short isoforms of p180. Positively charged amino acids are shown in red. Segments (amino acids 51-80) necessary for microtubule binding are underlined. g, Mapping of microtubule-binding domains of KTN1. Amino acid sequences around key microtubule-binding sites are shown at the bottom. Positively charged amino acids are in red. Segments (amino acids 112-120) necessary for microtubule binding are underlined. See Supplementary Information for uncropped western blots.