Figure 1.
Strategy for the identification of CD4-complexes in human primary Mφ.
CD14+ monocytes were isolated from human blood by magnetic cell sorting (MACS) and cultured for 7 days in the presence of M-CSF. One hundred million day 7 fully differentiated Mφ were left untreated (Condition 1, blue), treated with conditioned media from activated T cells (Induced CD4 internalization and degradation, Condition 2 red) or treated with conditioned media from activated T cells in the presence of the proteasomal inhibitor MG132 and the inhibitor of vacuolar ATPases bafilomycin (BafA1) (Induced CD4 internalization but blocked degradation, Condition 3 green). Eighteen hours later, cells were detached from tissue culture plates, lysed and large-scale anti-CD4 immunoprecipitations (IP) using monoclonal antibody against CD4 (clone QS4120) or isotype control IP were carried out. IP products were loaded onto SDS-PAGE pre-cast gels and electrophoresis were run. Protein gels were coomassie stained, gel lanes were cut into 10 equal pieces and trypsin-digested. Proteins were identified by LC-MS/MS.
Figure 2.
CD4 is internalized and degraded after treatment with conditioned media from activated T cells.
Mφ were treated with conditioned media from activated T cells for 18 hours or left untreated, followed by flow cytometry staining with directly conjugated mAb to CD4. A Black histogram represents the appropriate isotype control. Histograms show the intensity of the signal on the X-axis with a log10-scale and the percentage of maximum expression on the Y-axis. Representative staining of more than five donors tested (n>5). B Bars represent the mean percentage of Mφ expressing surface CD4 with SD error bars from ten independent donors (n = 10). C Total CD4 expression levels (surface + intracellular) were determined by dividing the geometrical MFI of the antibody staining over the MFI of the isotype control. Bars represent the mean values of five independent donors (n = 5) with SD error bars. In B and C, black bar corresponds to untreated Mφ and white bar corresponds to conditioned media treated Mφ (T cell Sup).
Figure 3.
Representative protein gels of anti-CD4 immunoprecipitations in Mφ.
Mφ were left untreated (Condition 1, blue), treated with conditioned media from activated T cells (Condition 2, red) or treated with conditioned media from activated T cells in the presence of 5 µM of MG132 and 100 nM of BafA1 (Condition 3, green). Eighteen hours later, cells were lysed and anti-CD4 immunoprecipitations were carried out. The final immunoisolates were resuspended in Laemmli sample buffer under reducing and denaturing conditions, before loading onto a SDS-PAGE pre-cast gel. Isotype control IgG immunoprecipitations were also performed to show non-specific background binding proteins.
Table 1.
Uniquely identified proteins in anti-CD4 co-immunoprecipitations in control Mφ (Condition 1).
Table 2.
Uniquely identified proteins in anti-CD4 co-immunoprecipitations in induced CD4 internalization and degradation in Mφ (Condition 2).
Table 3.
Uniquely identified proteins in anti-CD4 co-immunoprecipitations in induced CD4 internalization and blocked degradation in Mφ (Condition 3).
Table 4.
Proteins commonly identified in all conditions.
Figure 4.
Western blot analysis of CD4 co-immunoprecipitates in Mφ.
A total of 1×107 Mφ were left untreated (Condition 1, blue), treated for 18 hours with supernatants from activated T cells (Condition 2, red), treated for 18 hours with supernatants from activated T cells in the presence of 5 µM MG132 and 100 nM BafA1 (Condition 3, green), lysed and anti-CD4 immunoprecipitation reactions were carried out. Isotype control immunoprecipitations were also performed to show background protein binding. Immunoisolates were resuspended in Laemmli sample buffer under reducing and denaturing conditions and resolved on a SDS-PAGE gel. Membranes were incubated with antibodies against CD4, clathrin heavy chain (HC) 1, E3 Ubiquitin (Ub) ligase Itch, CD9 and CCR5. Primary antibodies were detected and scanned using the quantitative western blotting imaging Odyssey System. A representative blot of three different blood donors is shown (n = 3).
Figure 5.
Gene Ontology (GO) annotations of the uniquely identified proteins in anti-CD4 immunoprecipitations in Mφ.
Protein identifications from the three different conditions were exported from the in-house developed Central Proteomics Facilities data analysis pipeline (CPFP) and uploaded to ProteinCenter software. A illustrates the percentage of protein identifications versus protein cellular localizations (GO cellular annotations); B illustrates the percentage of protein identifications versus protein molecular functions (GO molecular annotations) and C illustrates the percentage of protein identifications versus protein biological functions (GO biological annotations). Blue bars represent the percentage of unique proteins identified in condition 1 (Resting macrophages); Red bars represent the percentage of unique proteins identified in condition 2 (Induced CD4 internalization and degradation); Green bars represent the percentage of unique proteins identified in condition 3 (Induced CD4 internalization and blocked degradation).