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. 2001 Oct;12(10):3031–3045. doi: 10.1091/mbc.12.10.3031

Figure 6.

Figure 6

Figure 6

Immunoblot analysis of reggie-1 and -2 in coimmunoprecipitates. Immunoprecipitation: ABs used for immunoprecipitation, as listed below each lane. Immunoblots of rat brain, DRGs, and PC12 cells (a–c), of astrocytes (c), and Jurkat cells (d) were probed with ABs: anti-reggie-1 mAB (a); anti-reggie-2 mAB and anti-fyn pAB (b); anti-caveolin-1 mAB, anti-reggie-1 mAB, and anti-reggie-2 mAB (c); and anti-reggie-1 mAB, anti-reggie-2 mAB, and anti-fyn pAB (d), respectively, as listed above the blots. Molecular weight markers are indicated to the left. (a) Reggie-1 is detected in immunoprecipitates with anti-reggie-2 and anti-fyn pABs from extracts of rat brain, DRGs, and PC12 cells. Coimmunoprecipitation of reggie-1 by anti-caveolin-1 pAB is not detectable. Anti-F3 pABs coprecipitate reggie-1 from extracts of rat brain and DRGs, but not from PC12 cells, which do not express F3. Anti-Thy-1 mAB (OX-7, coupled to Eupergit beads), coprecipitates reggie-1 from PC12 cells. Only a small amount of reggie-1 is seen after precipitation with an irrelevant AB (S4, coupled to the same Eupergit beads) due to nonspecific binding of reggie-1 mAB. Reggie-1 mAB (coupled to Dynabeads) precipitates reggie-1. Specific bands are marked by arrows. The bands above the specific reggie signal are due to the binding of secondary ABs to heavy chains of the precipitating ABs. (b) Reggie-2 is precipitated from rat brain extracts by anti-reggie-2 pAB, and, like reggie-1 (a), coprecipitates with F3 and fyn. Again, reggie-2 does not coprecipitate with caveolin-1 pAB. Reggie-2 mAB (coupled to Dynabeads) precipitates reggie-2 in PC12 cells (arrow). The bands above the specific reggie signal are due to the binding of secondary ABs to heavy chains of the precipitating ABs (control with secondary AB, 2nd only). Fyn mAB and Thy-1 mAB (OX-7, coupled to Eupergit beads) coprecipitates reggie-2 (arrows), but not the irrelevant AB S4 (on beads). Anti-fyn mAB (coupled to Dynabeads) precipitates fyn in PC12 cells and fyn is coprecipitated by anti-Thy-1 mAB (OX-7 on Eupergit beads). S4 on Eupergit beads as control. (c) Caveolin-1 is not detected after coimmunoprecipitation with anti-reggie-2 pAB or anti-F3 and anti-fyn pABs (rat brain), nor with anti-reggie-2 mAB (astrocytes), but is precipitated by anti-caveolin pAB. Anti-caveolin pAB and anti-reggie-2 mAB were coupled to Dynabeads. Anti-caveolin-1 pAB immunoprecipitates caveolin-1, but caveolin-1 is not coprecipitated by anti-reggie-2 or by anti-F3 or anti-fyn pABs. Reggie-1 is coimmunoprecipitated by anti-reggie-2 pAB and anti-fyn pAB in astrocytes, yet not by anti-caveolin pAB. Anti-caveolin pAB does not coprecipitate reggie-2. (d) In Jurkat cells, reggie-1 is coimmunoprecipitated by anti-reggie-2 pAB and mAB (arrow), by anti-fyn and anti-Thy-1 mABs (arrow) and is precipitated by anti-reggie-1 mAB. Preimmune sera (NS) and mouse IgG served as controls. All mABs were coupled to Dynabeads. Fyn, revealed by anti-fyn pAB in homogenates of Jurkat cells, is detected after coimmunoprecipitation with anti-Thy-1 mAB and anti-reggie-2 mAB (arrows) but no such bands appear after coprecipitation with nonspecific mouse IgG. Reggie-2 is coimmunoprecipitated by anti-fyn pAB and detected by anti-reggie-2 mAB.