Skip to main content
. 2016 Jun 20;213(6):679–692. doi: 10.1083/jcb.201601099

Figure 3.

Figure 3.

Nedd1/γ-TuRC and CDK5RAP2/γ-TuRC have different properties in vivo and in vitro. (A) Images of γ-tubulin in control proliferative keratinocytes and those with lentiviral-mediated knockdown of γ-tubulin, CDK5RAP2, or Nedd1. Arrows indicate centrosomes. Bar, 5 µm. (B) Quantification of centrosomal γ-tubulin levels in control, γ-tubulin KD, CDK5RAP2 KD, and Nedd1 KD cells (n ≥ 70 centrosomes from at least two independently derived cell lines for each condition). (C) Centrosomal MT nucleation rate in control, γ-tubulin KD, CDK5RAP2 KD, and Nedd1 KD cells (n ≥ 18 cells from at least two independent experiments). (D) Representative images of fields from MT assembly assays with tubulin alone, γ-TuRC alone, or purified γ-TuRCs incubated with the γ-tubulin binding domain of either Nedd1 or CDK5RAP2. Bar, 10 µm. (E) Quantification of MTs per field in the MT assembly assays, as indicated (n = 40 total random fields for each condition from two independent experiments). Data are presented as mean ± SEM. (F) γ-TuRCs purified by affinity for Nedd1 (NγTuRCs) or CDK5RAP2 (CγTuRCs) were used in MT assembly assays with addition of the γ-tubulin binding domain of CDK5RAP2, as indicated. Quantification of MTs per field in the MT assembly assays, as indicated (n = 15 random fields for each condition from three independent experiments). n.s., not significant; *, P < 0.05; ***, P < 0.001. Data are presented as mean ± SD.