Fig. 4: Constant tubulin biochemistry between the differentiation states.
From: Cell state-specific cytoplasmic density controls spindle architecture and scaling
a, Deconvoluted mass spectra of tubulins purified from ESCs (left) or DIF (right). Average masses of the most abundant signals and corresponding tubulin isoforms, βV (MW = 49,670.3 Da, UniProt P99024), βIVb (MW = 49,830.5 Da, UniProt P68372), βIIb (MW = 49,952.6 Da, UniProt Q9CWF2), αIb (MW = 50,151.1 Da, UniProt P05213). b, Mean relative abundances of the four most dominant isoforms purified from ESCs versus 96 h DIF (n = 3 biological replicates) measured by intact protein mass spectrometry. Error bars show s.e.m. Circles show replicates. Welch’s t-test (two-sided), αIb: P = 0.68, βIIb: P = 0.40, βIVb: P = 0.79, βV: P = 0.50. c, Tubulin PTMs are comparable between differentiation states. Western blots using whole-cell lysates and purified tubulin (n = 2 biological replicates). Affinity-purified Bos taurus (Bt) brain tubulin loaded as positive control. Poly-Glu, poly-glutamylation; K40-Ac, tubulin lysine 40 acetylation; Detyr, detyrosinated tubulin. d, Representative tubulin western blot of three differentiation time points and whole-cell lysates (n = 3 biological replicates) (top). For downstream calibration, defined masses of purified tubulin were blotted onto the same membrane (three technical replicates per batch of lysates). Coomassie-stained whole protein content on a replica gel (bottom). e, Tubulin consistently represents 1.5% of the cellular protein mass. Relative tubulin content in total cellular protein mass in whole-cell lysates of ECSs (0 h) versus 48 h or 96 h DIF cells. Bars show mean ± s.e.m. of n = 3 biological replicates. Circles show mean ± s.e.m. of three technical replicates per experiment. One-way analysis of variance (ANOVA), F-statistic = 0.1263, P = 0.88. NS, not significant, P > 0.05.
