Immunoprecipitation and Western blot analysis of patient lymphoblasts. (A) Western blot analysis of LMAN1 in lymphoblasts from probands in families B16 and B17 compared with cells carrying the MCFD2 I136T mutation and wild-type (wt) cells. (B) A small amount of LMAN1-MCFD2 complex is detected in lymphoblasts with the MCFD2 I136T mutation. Lysates from 5 × 106 cells were immunoprecipitated (IP) with rabbit anti-MCFD2 followed by Western blot analysis with monoclonal anti-LMAN1 (top panel) or anti-MCFD2 antibodies (bottom panel). (C) Lymphoblasts from the B17 proband were untreated (N) or treated with the proteasome inhibitors MG132 (MG) or lactacystin (Lac) for 6 hours. Lysates from 5 × 106 cells of B17 and B7 lymphoblasts were immunoprecipitated with monoclonal anti-LMAN1 followed by Western blot analysis with polyclonal rabbit anti-LMAN1.
