JCS062950 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Maturation of endogenous CD59, folate receptor and transferrin receptor in HeLa cells. Cells were pulsed with ³⁵S-methionine/cysteine for 10 minutes, chased for the indicated times and then subjected to immunoprecipitation with antibodies against CD59 (A), folate receptor (B) or transferrin receptor (C). Immunoprecipitates were treated (+) or not treated (−) with endo-H and separated on 15% (A), 12.5% (B) or 7.5% (C) SDS-polyacrylamide gels followed by autoradiography. Endo-H-resistant or partially endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular weights (kDa) on the left margin.

• Supplemental Figure S2 -

  Fig. S2. Effect of Sec24 isoform silencing on transport of VSV-G in HeLa cells. (A) Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. One day later, the cells were transfected with VSV-G cDNA, pulsed for 10 minutes with ³⁵S-methionine/cysteine, chased for 40 minutes, and subjected to immunoprecipitation with anti-VSV-G. Immunoprecipitates were treated with (+) or without (−) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H sensitive (arrowhead) forms of VSV-G are indicated on the right and molecular mass on the left margin. (B) Quantification of endo-H resistance of VSV-G from pulse-chase experiments illustrated in A (means ± s.d., n=3 independent experiments). Un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from control cells (P<0.05; Student�s t-test).

• Supplemental Figure S3 -

  Fig. S3. Efficiency of p23, p24 and p25 knockdowns in HeLa cells. (A) Cells were left untransfected (un), or were transfected with control siRNA (ctl) or siRNAs against p23 (23), p24 (24) or p25 (25). Three days later, NP40 cell lysates were analyzed by SDS-PAGE followed by immunoblotting using antibodies to p23, p24, p25 or tubulin (loading control). (B) Reduction of protein levels of p23, p24 and p25 by knockdown was quantified (means ± s.d., n=3). *Statistically significant difference from untransfected cells (P<0.05; Student�s t-test).

• Supplemental Figure S4 -

  Fig. S4. Tagged p24 and p23 properly interact with their endogenous counterparts. HeLa cells were transfected with Myc-p23 (A), transfected (+) or not (−) with HA-p24 (B). The cells were lysed in 1% Triton X-100 and immunoprecipitated with anti-p24 and anti-Myc or anti-HA. The immunoprecipitates were separated by SDS-PAGE followed by immunoblotting with antibodies against p24 and Myc (A) and HA and p23 (B). IgGL, light chain of immunoglobulins.

• Supplemental Figure S5 -

  Fig. S5. CD59 does not interact with p25. HeLa cells were transfected with Myc-p25 and subjected to immunoprecipitation with anti-CD59. The immunoprecipitate was separated by SDS-PAGE followed by immunoblotting with anti-CD59 and anti-Myc. Positions of molecular mass markers (kDa) are shown on the left margin.

• Supplemental Figure S6 -

  Fig. S6. Cholesterol depletion does not abolish the interaction of newly synthesized CD59 and p24 in raft and non-raft fractions. (A) Pooled lipid raft fractions (R, 15+20+25% of sucrose) and non-raft fraction (NR, 40% sucrose) from 15-minute pulse-labeled HA-p24-transfected cells were immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. (B) HA-p24-transfected HeLa cells were treated (+) or not (−) with 20 mM MβCD for 30 minutes, at 37°C, pulsed for 15 minutes, with ³⁵S-methionine/cysteine and subjected to immunoprecipitation with anti-CD59 followed by SDS-PAGE and autoradiography. Positions of molecular mass markers (kDa) are shown on the left margin.

• Supplemental Figure S7 -

  Fig. S7. Effect of MβCD-mediated cholesterol depletion on ER to Golgi transport of CD59, transferrin receptor and glycosylated retrieval-impaired Myc-ERGIC-53 (Myc-ERGIC-53). HeLa cells either untransfected (A and B) or transfected with the ERGIC-53 construct (C) were treated (+) or not (−) with 10 mM MβCD for 30 min at 37°C. Cells were pulsed with ³⁵S-methionine/cysteine for 10 min, chased for 30 min (for CD59) or 40 min (for transferrin-receptor and Myc-ERGIC-53), lysed in 1% Triton, and subjected to immunoprecipitation with antibodies against CD59, Myc or TfR. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, TfR and Myc-ERGIC-53 are indicated. MβCD: methyl-β-cyclodextrin. Positions of molecular weight markers (kDa) are shown on the left margin.