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Figure 2 | BMC Biochemistry

Figure 2

From: Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

Figure 2

ALIX is a POSH ubiquitination substrate. A. Overexpression of POSH induces ALIX poly-ubiquitination. Flag-ALIX and V5-POSH were expressed in H310 cells together with HA-ubiquitin. ALIX immune complexes were isolated and ALIX-ubiquitination was detected by Western blot with anti-HA. B. POSH ubiquitinates ALIX in vitro. Bacterially expressed ALIX (50 ng) and POSH (40 ng) were incubated in an in vitro ubiquitination reaction with the indicated Ubc's (E2's) as described in Materials and Methods. ALIX was subsequently isolated by immunoprecipitation with anti-FLAG and analyzed by Western blot with anti-FLAG. C. ALIX-linked ubiquitination is independent of K48 residue in ubiquitin. T7-tagged-ALIX was overexpressed in HEK-293 cells. ALIX immune complexes were subsequently isolated by immunoprecipitation with anti-T7 and incubated in an in vitro ubiquitination reaction with POSH and UbcH5c and with either native ubiquitin or UbK48R as described in Materials and Methods. Following the ubiquitination reaction, ALIX was isolated by immunoprecipitation with anti-FLAG followed by a stringent wash with buffer containing 0.6% SDS (to disrupt ALIX-POSH interaction and thus ensure detection of ALIX-conjugated ubiquitin only).

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