Journal: Molecular Biology of the Cell

Article Title: Physical and Functional Interaction of Transmembrane Thioredoxin-related Protein with Major Histocompatibility Complex Class I Heavy Chain: Redox-based Protein Quality Control and Its Potential Relevance to Immune Responses

doi: 10.1091/mbc.E09-05-0439

Figure Lengend Snippet: Association of TMX with calnexin. (A) Lysates (left) and anti-myc immunoprecipitates (right, IP: myc) from A549 cells stably transfected with control vector (V) or TMX-myc (TMX) were analyzed by immunoblotting (IB) with antibodies to calnexin (CNX), calreticulin (CRT), and myc. (B) 293 cells were subjected to immunoprecipitation with control (Ctrl) or anti-calnexin (CNX) antibody, and immunoblotted with antibodies to calnexin and TMX. (C) Top, schematic structure of TMX and its C-terminally truncated variants. SP, signal peptide; TRX domain, thioredoxin-like domain; TM, transmembrane domain. Bottom, 293 cells were transfected with different TMX-myc variants as indicated. Cell lysates (lanes 1–3) and anti-myc immunoprecipitates from the culture supernatants (lanes 4–6) were analyzed by immunoblotting with anti-myc antibody. (D) Left, TMX140-myc with (−) or without (+) ER-retention signal KDEL was transfected into 293 cells. The cells were fractionated into cytosolic (C) and membrane fractions (M), and the culture supernatants (S) were immunoprecipitated with anti-myc. The resulting samples were immunoblotted with anti-myc. Right, separation of cytosolic and membrane fractions was confirmed by immunoblotting with anti-thioredoxin (TRX, cytosolic marker) and anti-TMX (ER). (E) 293 cells transfected with control vector (V), TMX-myc (WT), TMX215-myc (215), TMX140-mycKDEL (140+), TMX/C56S·C59S-myc (CS), or TMX/C56A·C59A-myc (CA) were subjected to immunoprecipitation with anti-myc and immunoblotted with anti-CNX and anti-myc.

Article Snippet: Rabbit polyclonal antibodies specific for calreticulin (SPA-600), calnexin (SPA-865), and PDI (SPA-890) were from Assay Designs (Ann Arbor, MI).

Techniques: Stable Transfection, Transfection, Control, Plasmid Preparation, Western Blot, Immunoprecipitation, Membrane, Marker

Journal: Molecular Biology of the Cell

Article Title: Physical and Functional Interaction of Transmembrane Thioredoxin-related Protein with Major Histocompatibility Complex Class I Heavy Chain: Redox-based Protein Quality Control and Its Potential Relevance to Immune Responses

doi: 10.1091/mbc.E09-05-0439

Figure Lengend Snippet: Trapping mutants of TMX. (A) Lysates from 293 cells transfected with the indicated TMX-myc variants were separated under nonreducing (lanes 1–3) or reducing conditions (lanes 4–6) and immunoblotted with anti-myc. (B) top, 293 cells harboring the tetracycline-inducible TMX transgene (293:tet-TMX/P101T-myc) were treated with doxycycline (dox; 0–0.8 μg/ml) for 24 h. The expression of endogenous TMX and TMX/P101T-myc was examined by immunoblotting with anti-TMX antibody. Bottom, 293:tet-TMX/P101T-myc was treated with castanospermine (CST; 0, 0.5, and 1.0 mM) for 8 h and further incubated in the presence of dox (0.4 μg/ml) for 15 h. The formation of mixed disulfides was analyzed as described in A. (C) 293 cells were transfected with control vector, TMX/P101T-myc (P101T), or TMX/C56A·C59A-myc (C56·59A), and the lysates were immunoprecipitated with anti-calnexin (CNX) antibody. The immunoprecipitates were assessed for the presence of TMX-substrate complexes by immunoblotting with anti-myc. A putative TMX dimer is indicated by an asterisk.

Article Snippet: Rabbit polyclonal antibodies specific for calreticulin (SPA-600), calnexin (SPA-865), and PDI (SPA-890) were from Assay Designs (Ann Arbor, MI).

Techniques: Transfection, Expressing, Western Blot, Incubation, Control, Plasmid Preparation, Immunoprecipitation

Journal: Molecular Biology of the Cell

Article Title: Physical and Functional Interaction of Transmembrane Thioredoxin-related Protein with Major Histocompatibility Complex Class I Heavy Chain: Redox-based Protein Quality Control and Its Potential Relevance to Immune Responses

doi: 10.1091/mbc.E09-05-0439

Figure Lengend Snippet: TMX forms a disulfide-linked complex with MHC class I heavy chain. (A) 293 cells transfected with control vector or the indicated TMX-myc variants were subjected to immunoprecipitation with anti-myc. Cell lysates and the immunoprecipitates were analyzed by immunoblotting with anti-class I HC and anti-myc. Monomeric class I HC (open arrowhead) and the TMX-class I HC conjugate (closed arrowhead) are indicated (top). Red, reducing; Nonred, nonreducing. (B) Anti-myc immunoprecipitates from 293 cells transfected with TMX/C59A-myc were separated on a nonreducing/reducing two-dimensional gel, and analyzed by immunoblotting with antibodies to calnexin (CNX), class I HC, and myc. Calnexin was visible on the gel diagonal between the two arrows (circled). The arrowhead indicates MHC class I HC originated from the disulfide-linked complex.

Article Snippet: Rabbit polyclonal antibodies specific for calreticulin (SPA-600), calnexin (SPA-865), and PDI (SPA-890) were from Assay Designs (Ann Arbor, MI).

Techniques: Transfection, Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Two-Dimensional Gel Electrophoresis

Journal: Molecular Biology of the Cell

Article Title: Physical and Functional Interaction of Transmembrane Thioredoxin-related Protein with Major Histocompatibility Complex Class I Heavy Chain: Redox-based Protein Quality Control and Its Potential Relevance to Immune Responses

doi: 10.1091/mbc.E09-05-0439

Figure Lengend Snippet: Role of TMX in the degradation of MHC class I heavy chain. (A) A549 cells stably expressing TMX-myc were cultured in the absence (−) or presence (+) of tunicamycin (TM; 2 μg/ml) for 20 h. Cell lysates (lanes 1 and 2) and anti-myc immunoprecipitates (lanes 3 and 4) were analyzed by immunoblotting with anti-class I HC and anti-myc. Glycosylated (closed arrowhead) and nonglycosylated forms (open arrowhead) of class I HC are indicated. (B) A549 cells expressing TMX-myc were treated with or without tunicamycin as described in A. Cell lysates were first immunoprecipitated with anti-myc (1st IP), and the eluates were further immunoprecipitated with anti-calnexin (2nd IP). Coprecipitated class I HC was detected by immunoblotting (open arrowhead). *, coeluted antibody heavy chains (∼50 kDa). (C) 293 cells transfected with HLA-B27-V5 were incubated with (−) or without (+) MG-132 (10 μM) for 4 h and fractionated into cytosolic (C) and membrane (M) fractions. The translocation of MHC class I HC into the cytosol was examined by immunoblotting with anti-V5. The purity of each fraction was verified by immunoblots for ER marker calnexin. (D) 293 cells transfected with control vector (V) or HLA-B27-V5 (B27) were subjected to immunoprecipitation with anti-V5 and immunoblotted with anti-V5 and anti-TMX. Arrowhead denotes endogenous TMX coprecipitated with HLA-B27-V5. *, coeluted antibody light chains (∼25 kDa). (E) HLA-B27-V5 was cotransfected into 293 cells either with control vector (V) or with TMX-myc (TMX), and the translocation of HLA-B27 was analyzed as described in C. The gel was stained with Coomassie Blue (CBB) to ensure equal loading. Representative data are shown, and the band intensities of the cytosolic HLA-B27 are quantified. The Graph shows the mean ± SD of four experiments. (F) Control (Ctrl) and TMX knockdown cells (KD1 and KD2) were transfected with HLA-B27-V5, and the translocation of HLA-B27 was analyzed in the absence (−) or presence (+) of MG-132. Amido black staining of the same blot confirmed equal loading. Data are representative of two experiments and the intensities of HLA-B27 bands in the cytosol (lanes 4–6, open arrowhead) are quantified as shown in the graph.

Article Snippet: Rabbit polyclonal antibodies specific for calreticulin (SPA-600), calnexin (SPA-865), and PDI (SPA-890) were from Assay Designs (Ann Arbor, MI).

Techniques: Stable Transfection, Expressing, Cell Culture, Western Blot, Immunoprecipitation, Transfection, Incubation, Membrane, Translocation Assay, Marker, Control, Plasmid Preparation, Staining, Knockdown