Empty vectors pCMV-3??Flag and pGFP-N1 were used as a negative control. proteins, and is divided into two classes based on (Z)-9-Propenyladenine their expression patterns, the type I cancer-testis antigen and the type II ubiquitous MAGEs1,2. Both type I and type II MAGE proteins share a conserved domain known as the MAGE homology domain (MHD). Based on similarities in the MHD sequences and molecular sizes, the type II MAGE proteins can be divided further into two subgroups, Necdin subgroup and MAGE-D subgroup. Necdin subgroup consists of relatively short proteins ( 350 amino acid residues) whose MHD sequences are more homologous to that of Necdin. This subgroup includes Necdin, MAGE-F1, MAGE-G1, and MAGE-H1 in mammals. MAGE-D subgroup consists of larger proteins ( 650 amino acid residues), including MAGE-D1/NRAGE/Dlxin-1, MAGE-D2, MAGE-D3/Trophinin/Magphinin, MAGE-E1/MAGE-D4, and MAGE-L22,3. As one of homologous genes, gene (also designated immunoprecipitates. Right line, Flag-Mage-G1 protein was detected by anti-Flag antibody. immunoprecipitation; immunoblotting. (c) Identification of Flag-MAGE-G1 immunoprecipitated by anti-Flag beads in pre- or mixed cell lysates (1:1) by immunoblotting. immunoprecipitation; immunoblotting. SILAC Analysis Discriminates the Specific Binders from the Unspecific According to the stringent criteria for protein identification (see Materials and Methods), a total of 57 proteins were quantified with L/H ratios (Fig. 2 and Supplementary Table 1). Using significance B value (p? ?0.05) as the threshold to distinguish the specific MAGE-G1-interacting proteins14, 27 proteins were demonstrated as having significant abundance changes (L/H ratios? ?1.70 BL21 were incubated with Flag-MAGE-G1 expressed in HEK293T, and precipitated by Glutathione-Sepharose 4B beads. The result showed that Flag-MAGE-G1 was detected in the GST-FSCN1 precipitate (Fig. 4a). The expression vectors that encoded MAGE-G1 fused with a Flag-tag (pCMV-3??Flag-fused with a GFP-tag (pGFP-N1-and GFP-(Fig. 4b). Reciprocal assays showed that Flag-MAGE-G1 was detected in anti-GFP immunoprecipitate from COS-7 cells co-transfected with Flag-and GFP-(Fig. 4c). For negative control, there was no GFP-FSCN1 or Flag-MAGE-G1 detected in anti-Flag or anti-GFP antibody immunoprecipitates from cells co-transfected with Flag-and GFP-BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-FSCN1 were detected (Z)-9-Propenyladenine with indicated antibody. Full-length blots are included (Z)-9-Propenyladenine in a Supplementary Information. (b) COS-7 cells were co-transfected with either Flag-plus GFP-or pCMV-3??Flag empty vector plus pEGFP-expression plasmids. 25?g of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-FSCN1 (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. (c) COS-7 cells were co-transfected with either Flag-plus GFP-or pEGFP empty vector plus (Z)-9-Propenyladenine Flag-expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. (d) Whole cell lysates from RA-treated P19 cells were immunoprecipitated with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation (IP) and 25?g whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-FSCN1 antibody. To further confirm the interaction between MAGE-G1 and FSCN1 during neuronal differentiation, we endogenous immunoprecipitation was performed in P19 cells after treated with RA for 6 days. Results showed that endogenous FSCN1 was co- immunoprecipited with endogenous MAGE-G1 by anti-MAGE-G1 antibody in P19 cells after 6-day treatment with RA s, but not by control IgG (Fig. Rabbit polyclonal to ACMSD 4d). The fidelity of anti-MAGE-G1 antibody (B-Bridge, USA) and anti-FSCN1 antibody (Sigma-Aldrich, USA) used in endogenous immunoprecipitation experiments was confirmed (Supplementary Figure 3a and b). Validation of the Interaction between MAGE-G1 and VIME To confirm the interaction between MAGE-G1 and VIME, we analyzed the association of these two proteins by GST pull-down assay in bacteria and by immunoprecipitation in transfected mammalian cells. GST or GST-VIME expressed in BL21 were incubated with Flag-MAGE-G1 expressed in HEK293T, and precipitated by Glutathione-Sepharose 4B beads. The result showed that Flag-MAGE-G1 were detected respectively in the GST-VIME precipitate (Fig. 5a). Expression vectors Flag-and GFP-were constructed and transiently co-transfected into COS-7 cells. Empty vectors pCMV-3??Flag and pGFP-N1 were used as a negative control. Immunoprecipitation and immunoblotting analysis were performed on cell lysates from those transfected cells. The result showed that GFP-VIME was detected in the anti-Flag immunoprecipitate from COS-7 cells co-transfected with Flag-and GFP-(Fig. 5b). Similarly, Flag-MAGE-G1 was detected in the anti-GFP immunoprecipitate from COS-7 cells co-transfected with Flag-and GFP-(Fig. 5c). Open in a separate window Figure 5 Validation of the interaction between MAGE-G1 and VIME by GST pull-down and co-immunoprecipitation experiments.(a) GST or GST-VIME proteins.