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. 2022 Feb 26;298(4):101778. doi: 10.1016/j.jbc.2022.101778

Figure 6.

Figure 6

c-Abl-mediated γ-tubulin phosphorylation regulates MT nucleation.A, immunofluorescence microscopy of wild-type or abl1−/−abl2−/− U2OS cells stained with anti-α-tubulin (green) and anti-PCNT (red). B, U2OS cells treated with nilotinib (5 μM) or DMSO for 7 days were stained with anti-α-tubulin (green) and anti-PCNT (red). C, abl1−/−abl2−/− U2OS cells were infected with adenovirus expressing Myc-c-Abl or Myc-c-Abl(K290R) and then stained with anti-α-tubulin (green) and anti-Myc (red). D, γ-tubulin-Flag or γ-tubulin(Y443F)-Flag U2OS cells in which endogenous expression was knocked down by RNAi (red) were stained with anti-α-tubulin (green) and anti-PCNT (purple). E, MCF-7 cells treated with nilotinib (5 μM) or DMSO for 7 h were incubated on ice for 2 h to depolymerize the MTs and then immersed in DMEM at 37 °C immediately. The cells were fixed at the indicated time points and stained with anti-α-tubulin. F, U2OS cells stably expressing γ-tubulin-Flag and γ-tubulin(Y443)-Flag were incubated on ice for 1 h and then analyzed as in (E). G, cells in (F) and treated as in (F), 5 min after 37 °C incubation, were fixed and stained with anti-α-tubulin (green) and anti-GM130 (red). The focal planes of centrosomal MTs are shown in the last panels marked with Z2. H, Wild-type or abl1−/−abl2−/− U2OS cells were infected with Myc-c-Abl- or Myc-c-Abl(K290R)-expressing adenovirus and stained with anti-α-tubulin (green) and/or anti-Myc (red).