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Figure S3. Zeta-tubulin precipitation reveals TRiC/CCT and GFP-zetatubulin is cytoplasmic in frog and mouse tissue culture cells. Related to Figures 2 and 4D. A) GFP nanobody-conjugated beads were used to affinity purify GFP-zetatubulin from GFP-zeta-tubulin stable cells, and GFP nanobody-conjugated beads were incubated separately with A6 cell lysate as a control. The most abundant co-purifying proteins as identified by mass spectrometry are shown, relative to their respective peptide counts. Hits include zeta-tubulin (blue), all 8 subunits of
TRiC/CCT (green), heat shock or protein folding factors (pink), and proteasome associated proteins (yellow). B) Wild-type A6 cells and the GFP-zeta-tubulin stable line were fixed in methanol and stained for GFP (green), gamma-tubulin (red), and DAPI (blue). GFP was only detected in the stable cells, and GFP-zetatubulin labels cytoplasm and does not co-localize with centrosomes. Scale bar, 5 µm. C) 3T3 mouse fibroblasts were infected with GFP-zeta-tubulin lentiviruses and fixed in methanol. Cells were stained for GFP (green), poly-glutamylated tubulin (red), and DAPI (blue). Inset shows a magnified image of the cilium (3x). Zeta-tubulin does not localize to microtubule-based structures in mouse cycling cells. Scale bar, 5 µm. |
