Skip to main content
. Author manuscript; available in PMC: 2008 Aug 14.
Published in final edited form as: Cancer Res. 2006 Feb 1;66(3):1702–1711. doi: 10.1158/0008-5472.CAN-05-3092

Figure 2. Detection of PERK and eIF2α phosphorylation.

Figure 2

A, T-Hep3 and D-HEp3 cells were treated for the indicated time points with 5 μg/mL tunicamycin (Tn) and then lysed with radioimmunoprecipitation assay lysis buffer and analyzed by Western blot for phospho-Thr980 (p-Thr980)-PERK and for PERK, which served as a loading control. Absorbance (O.D.) numbers are indicated below the PERK blot and are the result of normalizing phospho-PERK (p-PERK) to PERK.B, control and tunicamycin-treated cells were lysed with radioimmunoprecipitation assay lysis buffer and analyzed by Western blot for phospho-Ser51 (p-Ser51)-eIF2α and eIF2α levels, which served as a loading control. C, lysates prepared with radioimmunoprecipitation assay lysis buffer were Western blotted for phospho-PERK from the indicated cell lines (top) or for phospho-PERK and ERK (middle and bottom) in D-HEp3 cells treated with or without 5 μmol/L SB203580 for 24 hours. ERK was used as a loading control. T, T-HEp3; D-neo and Ddnp38, D-Hep3 cells stably expressing an “empty” vector or a vector encoding a dominant-negative p38α. D and E, T-Hep3 and D-HEp3 cells were transiently cotransfected with the GADD153-EGFP reporter plasmid and the indicated plasmids (GADD34 or PERKΔC) (D) or were transfected with pEGFP alone (E). GADD153-induced EGFP was analyzed 48 hours after transfection by fluorescence microscopy (D, left) or FACS, where a total 5 × 104 events were captured and the GFP fluorescence in FLH2>10 was plotted in a histogram (D, right). Constitutive EGFP expression was also analyzed by fluorescence microscopy as before, and the percentage of EGFP-expressing cells in FLH2>10 was plotted (D) Bar, 180 μm. F, cells cotransfected as in (D) with DNp38 or MKK6Eb plasmids were processed for FACS analysis. Columns, mean number of GFP-positive events in FLH2>10; bars, SD.