Figure 5
Western analyses of the dynactin complex in the ∆capA mutant. (A) Western blots showing that normal amounts of dynein HC and Arp1 are pulled down with p150-GFP in the ∆capA mutant extract. Cells were cultured for overnight at 37 °C in MM + glucose medium. A strain without any GFP tag was used as a negative control. Another control is the strain containing GFP-dynein HC in the alcA-Arp1 background, in which the expression of Arp1 was repressed by glucose and the stability of p150 is decreased6. Cropped pieces with black outlines indicate blots probed by different antibodies against the indicated proteins (see Supplemental Fig. 10 for the original blots). (B) Western blots showing that dynein HC, dynactin p150 and Arp1 are pulled down with ∆C-HookA-GFP in the ∆capA mutant extract. The antibody against GFP (from Clontech) has been used previously29. The affinity-purified antibodies against dynein HC, dynactin p150 and Arp1 have been described and used previously6,40. Cropped pieces with black outlines indicate blots probed by different antibodies against the indicated proteins (see Supplemental Fig. 11 for the original blots). (C) A quantitative analysis on the ratio of pulled-down p150, Arp1 or dynein HC to ∆C-HookA-GFP as well as the ratio of pulled-down dynein HC or Arp1 to p150. The values were generated from western analyses (shown in B) of three independent pull-down experiments. The wild-type values are set as 1. Scatter plots with mean values were generated by using Prism 7. For all the ratios, there is no significant difference between wild-type and ∆capA at the 95% confidence level based on nonparametric tests without assuming any information on the distribution (p = 0.1 for Arp1/∆C-HookA, p = 0.7 for p150/∆C-HookA, p = 0.7 for dynein HC/∆C-HookA, p = 0.1 for dynein HC/p150 and p = 0.7 for Arp1/p150, two-tailed) (unpaired, Mann-Whitney test, Prism 7).
