Figure 6. Overexpression of SAS-1 in human cells reveals microtubule binding and stabilization activities.

(A–R) U2OS cells not expressing (control, A, E, I, M) or expressing wild type SAS-1-GFP (B, F, J, N, Q), SAS-1-P419S GFP (C, G, K, O, R) or SAS-1-G452E GFP (D, H, L, P) stained for GFP (magenta), as well as α-tubulin (A–D), acetylated tubulin (E–P) or centrin (Q–R) (yellow). DNA is in cyan. Insets in (A–P) are 2 fold magnified views, those in (Q–R) 3 fold magnified views. In (M–R), microtubules were depolymerized by placing cells 30 min on ice cold lead blocks, in (I–L), by treating them with 1 µM nocodazole for 1 h. (S) Immunoprecipitation using the GFP nanotrap of the indicated cell lysates probed with the indicated antibodies. In  =  Input  = 140 µg, FT  =  flow through  = 140 µg, IP  =  immunoprecipitation  = 20% of input. We used 2 mg of protein lysate for GFP, 10 mg for SAS-1-P419S-GFP, and 22 mg for SAS-1-GFP to obtain sufficient material. The tubulin and acetylated tubulin intensity was normalized with the SAS-1 band in SAS-1-GFP and SAS-1-P419S-GFP, and with the GFP band for GFP. Note that the upper blot is from a different membrane than the lower one. The asterisk indicates a probably dimeric form of GFP. N = 3 for the wild type and the mutant; representative blots are shown.