FIG 4 .

PKR undergoes K63 and K48 ubiquitination in response to LPS. (A) HEK293T cells were transfected with cDNA constructs expressing V5-tagged wild-type (WT) PKR and/or HA-tagged WT ubiquitin, HA K63-only Ub, or HA K48-only Ub. Twenty-four hours later, cells were lysed, and PKR was immunoprecipitated from whole-cell lysates. Immunoprecipitates were subjected to SDS-PAGE and probed with monoclonal antibody against HA. (B) Raw 264.7 cells were incubated overnight with medium alone or 100 ng/ml of LPS. Cells were washed and restimulated with 250 ng/ml LPS for the indicated times. Immunoprecipitation was carried out with anti-K63 Ub chain monoclonal antibody. (C and D) MAT4 cells were transfected with cDNA constructs expressing either K63-only HA-tagged Ub (C) or K48-only HA-tagged Ub (D) and immediately treated overnight with medium alone or 100 ng/ml LPS. Eighteen hours following primary treatment, cells were washed and stimulated with 250 ng/ml LPS, PKR was immunoprecipitated from whole-cell lysates, and immunoprecipitates were subjected to SDS-PAGE, followed by probing with anti-HA monoclonal antibody. (E) Naive (N) or LPS-tolerized (T) MAT4 cells were restimulated with 250 ng/ml LPS, and phospho-ERK levels were assayed by Western blotting. These data are representative of 4 independent experiments.