Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc-0

D with Cosmc-HA or with T-synthase–HPC4 and stained with rat anti-HA IgG1 (green) and rabbit anti-calnexin IgG (red; A–C; bars, 4 μm) or with mouse anti-HPC4 (green) and rabbit anti–α-ManII (red; D–F; bars, 8 μm). (G and H) Sucrose gradient subcellular fractionation. 293T cells transiently transfected with HPC4-Cosmc were harvested and homogenized. The postnuclear supernatant (PNS) was loaded onto a sucrose gradient. After ultracentrifugation, 16 fractions (∼0.6 ml/fraction) were collected and measured for both T-synthase and β4–Gal-T activity (G) and analyzed on Western Blot with anti-HPC4 and anti-KDEL (H).<p><b>Copyright information:</b></p><p>Taken from "Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc"</p><p></p><p>The Journal of Cell Biology 2008;182(3):531-542.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500138.</p><p></p

Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc-2

Ld-type Cosmc. Cells for Cosmc-HA and media from cell coexpressing HPC4–sT-synthase and wild-type Cosmc were harvested. The cell extracts and media were loaded on ATP-Sepharose column for chromatography and eluted with 50 mM ATP, respectively. The washes and eluates were analyzed by Western blot with anti-HA for Cosmc and anti-HPC4 for T-synthase, as indicated. (B) HPC4-sCosmc and HPC4–sT-synthase (coexpressed with wtCosmc) were expressed in Hi-5 cells and purified from the media. 3 μg of Cosmc and T-synthase were photolabeled with 8-Azido α-[P]ATP, analyzed on SDS-PAGE, and transferred to nitrocellulose membrane for radioautogram and Western blot with anti-HPC4. Black line indicates that intervening lanes have been spliced out.<p><b>Copyright information:</b></p><p>Taken from "Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc"</p><p></p><p>The Journal of Cell Biology 2008;182(3):531-542.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500138.</p><p></p

T-synthase–HPC4 expressed in LSC and LSB cells treated with 10 μM lactacystin was affinity purified and analyzed on SDS-PAGE under reducing and nonreducing conditions and Western blotted with anti-HPC4 (A)

Cell extracts were made and cross-linking was performed by adding 5 mM DSS. The recombinant T-synthase was purified and analyzed by Western blotting under reducing conditions (B). Sf-9 cells infected with Baculovirus-expressing T-synthase-HPC4 or coinfected with Baculovirus encoding wtCosmc were harvested and one portion was made for cell extracts. The cell extracts and the other portion of intact cells were treated with DSS. T-synthase was purified from the cell extracts and analyzed by Western blot with anti-HPC4 (C). Black line indicates that intervening lanes have been spliced out.<p><b>Copyright information:</b></p><p>Taken from "Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc"</p><p></p><p>The Journal of Cell Biology 2008;182(3):531-542.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500138.</p><p></p

(A and B) MG-132 causes accumulation of full-length T-synthase protein in LSC cells but failed to restore its activity

LSC and LSB cells stably expressing T-synthase–HPC4 were treated with 10 μM MG-132 for 12 h and harvested. Cell extract was made and one portion was used for measuring T-synthase activity (A), whereas the other portion was analyzed on SDS-PAGE (30 μg protein/lane) by Western blot with anti-HPC4 (B). Error bars represent ±1 SD from the mean. Black lines indicates that intervening lanes have been spliced out. (C–H) T-synthase expressed in LSC cells resides mainly in heavy membrane fractions (RER), whereas T-synthase from LSB cells is in light membrane fractions (Golgi). LSC and LSB cells as in A and B treated with 10 μM lactacystin were homogenized and cell homogenate was fractionated on a sucrose gradient by ultracentrifugation. The fractions were collected and the activities of β4–Gal-T and T-synthase in fractions were determined (C). The fractions were also analyzed by Western blotting with anti-HPC4 (D–F), anti-20S proteasome α1-subunit (G), and anti-KDEL (GRP78 and GRP94; H). In F, lane 2 represents the combination of fractions 1 and 2. (I–O) T-synthase expressed in LSC cells resides mainly in the lumen of the RER and was associated with GRP78. The cell PNS of LSC and LSB cells as in A and B was digested with 40 μg/ml trypsin in the presence or absence of 0.2% Triton X-100, and T-synthase was analyzed under reducing SDS-PAGE by Western blotting with HPC4 mAb (I and J). As controls, calnexin and 20S proteasome α1-subunit were also analyzed by Western blotting with their respective antibody (K and L). T-synthase was purified from LSC and LSB cells and Western blotted with anti-HPC4 (M) and anti-KDEL (GRP78; N). GRP78 in the cell extract corresponding to one-tenth of the material was detected to confirm comparable starting amounts of material (O).<p><b>Copyright information:</b></p><p>Taken from "Regulation of protein O-glycosylation by the endoplasmic reticulum–localized molecular chaperone Cosmc"</p><p></p><p>The Journal of Cell Biology 2008;182(3):531-542.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500138.</p><p></p