Skip to main content
. Author manuscript; available in PMC: 2017 Feb 8.
Published in final edited form as: Dev Cell. 2016 Feb 8;36(3):331–343. doi: 10.1016/j.devcel.2016.01.001

Figure 4. The S718-S721 phosphorylation relieves the intramolecular interaction of ALIX in mitotic cells.

Figure 4

(A) Left: IB of dIE and dME with MPM2. Middle: dIP with #4381, followed by IB with 3A9. Right: dIP with 3A9, followed by IB with 3A9 and αpS2. (B) dIP of IE and ME from cells ectopically expressing WT or S2A GFP-ALIX with #4381, followed by IB with αGFP. (C) dIP of the IE and ME samples in (B) with αGFP, followed by IB with αGFP and αpS2. (D) Left: IB of IE and ME from cells ectopically expressing indicated forms of GFP-ALIX with the indicated antibodies. Right: IP of the IE or ME samples with IgG or the indicated α-ALIX, followed by IB with the indicated antibodies. (E) IP of ME from cells ectopically expressing the indicated forms of GFP-ALIX with IgG or the indicated α-ALIX, followed by IB of input proteins and immunocomplexes with αGFP. (F) IP of IE from cells ectopically coexpressing FLAG-TSG101 and GFP or the indicated forms of GFP-ALIX with αFLAG in the presence of 1% Triton X-100, followed by IB of input proteins and immunocomplexes with the indicated antibodies. (G) Left: experimental flowchart for cell treatments. Middle: IB of IE and ME from collected cells with MPM2. Right: determination of the S718-S721 phosphorylation by dIP with 3A9, followed by IB with 3A9 and αpS2 (n = 3 ± SD). See also Figure S2.