JCS046979 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Detergent insolubility of PKCι is not dependent upon lipid raft association. At 10 days after seeding, CACO-2 cells were washed in PBS and extracted in Triton X-100 and spun (first supernatant, S1). Then, the pellet was extracted in 1.5 M KCl (second supernatant, S2 and pellet, P). Ponceau-S staining is shown as a loading control. (A) An extensively used marker of lipid rafts, ganglioside GM1, is present only in the first supernatant (S1) fraction of CACO-2 cells. Aliquots of each diluted fraction (S1, S2 and P) equivalent to 10 and 4 µg of protein, respectively, were dot-blotted onto nitrocellulose, incubated with 210 ng/mL HRP-conjugated cholera toxin, and processed for chemiluminescence detection. (B) Treatment of CACO-2 cells with cholesterol sequestering agents 10mM β-cyclodextrin (CD) (6 hours) or 10 µg/ml filipin III (1 hour) does not influence the presence of PKCι in highly insoluble fractions of CACO-2 cells (P control, P CD, P filipin) together with IFs. M_r of standards are in kDa. GM1 was assayed as described by others (Pang et al., 2004). CACO-2 cells were extracted and fractionated (S1, S2 and P) as described above. Next, 10 µL of each diluted fraction (S1, S2 and P) equivalent to 10 and 4 µg of protein were dot-blotted in triplicates onto nitrocellulose, incubated with 210 ng/mL HRP-conjugated cholera toxin (List Biological Laboratories), and processed for chemiluminiscence detection. For PKCι detection in the P fraction, CACO-2 cells were incubated with 10mM of β-cyclodextrin (CD) for 6 hours or with 10 µg/ml of filipin III for 1 hour at 37°C. Control cells were incubated with DMSO only.

• Supplemental Figure S2 -

  Fig. S2. (A) Silver-stained 2D-gel showed the purity of the keratin preparation (IF). A total of 10 µg keratins purified from CACO-2 cells were loaded. The pH gradient (horizontal axis) was 1-13. The sensitivity of the method varies with the proteins but minimum detectable concentrations are typically below 1 ng/mm² for most proteins (Oakley et al., 1980). (B) A 1 µg sample of recombinant Hsp70 (Stressgen) run in conventional SDS-PAGE stained by silver stain. M_r of the standards are in kDa.

• Supplemental Figure S3 -

  Fig. S3. PKCι undergoes constitutive proteasomal degradation. (A) CACO-2 cells were cultured for 7 days, and then incubated with 0 (only DMSO vehicle, contr), 10, or 20 µM proteasome inhibitor MG-132 for 6 hours. Then, SDS extracts were analyzed by PAGE and immunoblot using anti-PKCι antibody, anti-ubiquitinylated protein antibody, and anti-actin antibody (loading control). M_r of standards are in kDa. (B) The relative amount of PKCι and ubiquitinylated proteins respect to the untreated control (contr) were obtained after normalizing the bands to the amount of actin. Data represent the mean ± s.d. of three independent experiments.