Table 1. Claudin-5 partners in hCMEC/D3 cells identified by mass spectrometry.
| Protein name | SWISSPROT access number | Cellular compartment | MW (Da) | Mascot score | Queries |
|---|---|---|---|---|---|
| p120 catenin (*) | O60716 | cyt., pl. mb, nucl. | 108,674 | 1,272 | 22 |
| Guanine nucleotide-binding protein Gi subunit α2 (Gαi2) (*) | P04899 | pl. mb | 40,995 | 728 | 8 |
| Actin (β-actin) (*) | P60709 | cyt. | 42,052 | 282 | 5 |
| HSPA5/BiP (*) | P11021 | cyt. | 72,402 | 200 | 4 |
| Myosin-Ic | O00159 | cyt. | 121,724 | 132 | 4 |
| Stomatin | P27105 | pl. mb. | 31,882 | 174 | 3 |
| Histone H4 | P62805 | nucl. | 11,360 | 140 | 3 |
| VE-cadherin (*) | P33151 | pl. mb. | 87,516 | 122 | 3 |
| HSPA9 | P38646 | cyt. | 73,920 | 121 | 3 |
| Claudin-5 (*) | O00501 | pl. mb. | 23,816 | 154 | 2 |
| Junction plakoglobin, γCatenin (*) | P14923 | pl. mb. | 82,434 | 88 | 2 |
| Caveolin-1 (*) | Q03135 | pl. mb, caveolae | 20,472 | 56 | 1 |
| PKCθ (*) | Q04759 | cyt. | 83,407 | 35 | 1 |
Pl. mb., plasma membrane; cyt., cytosol; nucl., nucleus.
Caveolae fractions were isolated as described in Materials and methods from hCMEC/D3 cells, either untreated or treated with 1 M mannitol for 30 minutes or treated with 1 M mannitol plus a 48-hour recovery period. Claudin-5 was immunoprecipitated with anti-claudin-5 polyclonal antibodies. Coimmunoprecipitated proteins were identified by nano-LC/MS/MS analysis. Each protein was identified in at least three independent experiments. Are indicated: SwissProt access numbers, Mascot scores (scores >35 with P<0.01), queries (number of sequenced peptides identified). Cellular compartments are indicated according to the SwissProt data bank. The table presents data from one out of three independent mass spectrometry analyses. Asterisks (*) indicate proteins known to be localized in caveolae and/or at cell–cell junctions. Proteins in bold (Gαi2, β-actin, and PKCθ) were detected in control and postrecovery conditions, but not detected after 1 M mannitol treatment.