Figure 3. Effects of ABL-knockdown, mAbl-reconstitution and lysosomal inhibitors on ubiquitinated caspase-8.

a. Inducible vs. constitutive levels of Ub-C8 in parental vs. ABL-knockdown HT29 cells. Following treatment with 2.0 µg/ml Dox and/or 200 ng/ml Tr for 6 hr, cells were lysed in 1% SDS, boiled, immunoprecipitated with anti-caspase-8 and probed with anti-Ub (see Materials & Methods). IgG: anti-caspase-8 antibody added to the lysis buffer and signals on the blot are from the antibody itself. WCL: whole cell lysates. Note that caspase-8 was mostly not ubiquitinated as the increase in Ub-C8 did not significantly reduce the intensity of the 55kd pro-caspase-8 band in WCL, and that Ub-C8 was only detected in the anti-C8 immunoprecipitates. Poly-Ub levels in WCL were not affected by the treatments with Dox and/or TRAIL or by ABL-knockdown.

b. Reduction of Ub-C8 levels in ABL-knockdown cells through mAbl-reconstitution. Ub-C8 was measured as in (a). Reconstitution with WT- or µNES-mAbl reduced Ub-C8 in the ABL-knockdown cells. Reconstitution with KD or µNLS-mAbl did not reduce Ub-C8. IgG as in a.

c. Effects of Bafilomycin A1 (BAF) and MG132 on Ub-C8. Cells were treated with 200 nM BAF or 20 µM MG132 for the indicated time (hr) and Ub-C8 measured as in (a). Veh: vehicle (DMSO). Note that increases in WCL poly-Ub were similar between the parental and the ABL-knockdown HT29 cells. IgG as in a.

d. Effects of Chloroquine (CQ) and MG132 on Ub-C8. Cells were treated with 10 µM chloroquine (CQ) and/or 20 µM MG132 for the indicated time (hr) and Ub-C8 measured as in (a). IgG as in a.

e. Effects of BAF (200 nM) on the levels of p62/SQTM1, LC3, and LC3II at the indicated time (hr). Veh: vehicle (DMSO). BAF treatment caused similar increases in p62 and LC3II in the parental and the ABL-knockdown HT29 cells.