Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Unfolded Protein Response by eIF2B? Isoforms

doi: 10.1074/jbc.M110.153148

Figure Lengend Snippet: A distinct eIF2Bδ isoform is preferentially recognized by a monoclonal antibody. A, protein from several cell lines with inactive and active UPRs was immunblotted for eIF2Bδ using a monoclonal antibody as well as a polyclonal antibody directed against the C terminus. eIF2α is shown as a loading control. B, two isoforms, derived from alternative splicing of the eIF2Bδ mRNA, result in peptides with divergent N-terminal sequences. C, overexpression of V2 and V1 eIF2Bδ isoforms demonstrates that the V1 form is recognized by a monoclonal antibody, whereas both forms are recognized by a polyclonal antibody raised against the C-terminal portion of human eIF2Bδ.

Article Snippet: Interactions of eIF2α and eIF2β were assessed with HA-tagged eIF2β and non-tagged eIF2α (provided by H. Harding), and interactions with eIF2Bβ and eIF2Bδ were done by first immunoprecipitating endogenous eIF2Bβ with antibody (H300, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) attached to fast flow protein A-Sepharose beads (Amersham Biosciences) and then immunoblotting with eIF2Bδ antibody.

Techniques: Derivative Assay, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Unfolded Protein Response by eIF2B? Isoforms

doi: 10.1074/jbc.M110.153148

Figure Lengend Snippet: Cells with an inactive UPR express the V2 isoform of eIF2Bδ. A, a panel of cell lines that had previously been classified for UPR activation (Fig. 1) were assessed for eIF2Bδ isoform expression with both the polyclonal antibody that recognizes both variants of eIF2Bδ protein and the monoclonal antibody that does not recognize the V2 form of eIF2Bδ. eIF2α expression is demonstrated as a loading control. B, the result of three separate immunoblots were quantitated as described under “Materials and Methods,” and the ratio of expression as determined by the monoclonal antibody to expression as determined by the polyclonal antibody is displayed in group I and group II cells (left). In addition, the average ± S.E. (error bars) of polyclonal and monoclonal anti-eIF2Bδ signal in group I and group II cells is displayed (right). *, p < 0.05 versus group I cells. C, PCR primers were designed to amplify either both isoforms of eIF2Bδ (common), only V1, or only V2. Specificity is demonstrated by utilizing cDNAs for each isoform. D, PCR primers were used to assess the expression of eIF2Bδ mRNA isoforms of group I and group II cells (left), along with average expression in group I and group II cells (with S.E. and p values by Student's t test).

Article Snippet: Interactions of eIF2α and eIF2β were assessed with HA-tagged eIF2β and non-tagged eIF2α (provided by H. Harding), and interactions with eIF2Bβ and eIF2Bδ were done by first immunoprecipitating endogenous eIF2Bβ with antibody (H300, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) attached to fast flow protein A-Sepharose beads (Amersham Biosciences) and then immunoblotting with eIF2Bδ antibody.

Techniques: Activation Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Unfolded Protein Response by eIF2B? Isoforms

doi: 10.1074/jbc.M110.153148

Figure Lengend Snippet: Generation of cells that only express one eIF2Bδ isoform. A, U2OS cells were infected either with scramble (scr) or eIF2Bδ shRNAs, and protein expression for eIF2Bδ was determined. B, shRNA 1 (sh#1) from A (sequence defined under “Materials and Methods”) was used to generate eIF2Bδ knockdown U2OS cells, which were then either infected with retroviral empty vector (pQCXIN) or retrovirus expressing the V2 or V1 isoforms. Immunoblot (top) and quantitation (below) are displayed.

Article Snippet: Interactions of eIF2α and eIF2β were assessed with HA-tagged eIF2β and non-tagged eIF2α (provided by H. Harding), and interactions with eIF2Bβ and eIF2Bδ were done by first immunoprecipitating endogenous eIF2Bβ with antibody (H300, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) attached to fast flow protein A-Sepharose beads (Amersham Biosciences) and then immunoblotting with eIF2Bδ antibody.

Techniques: Infection, Expressing, shRNA, Sequencing, Plasmid Preparation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Unfolded Protein Response by eIF2B? Isoforms

doi: 10.1074/jbc.M110.153148

Figure Lengend Snippet: Expression of eIF2Bδ isoforms regulates the unfolded protein response. A, U2OS control cells and U2OS cells expressing either the V1 or V2 eIF2Bδ isoform (as described in Fig. 4) were treated with tunicamycin (Tm) (2.5 μg/ml) for various times, after which protein was isolated for immunoblots (A), which were then quantitated (B) for proteins up-regulated during UPR activation. A representative immunoblot is displayed along with quantitation of 2–4 immunoblots and S.E. (error bars). C, mRNA was collected for real-time PCR for two ATF-4 targets up-regulated during the UPR. D, 293 cells were generated with endogenous eIF2Bδ knocked down and expressing either eIF2Bδ V2 or V1 resistant to knockdown as in Fig. 4. E, 293 cells, as described in D, were treated with tunicamycin for the described times and immunoblotted for ATF-4 and its target ATF-3. F, U2OS cells expressing control vectors or eIF2Bδ isoforms were treated with tunicamycin, and RNA was collected to assess XBP-1 splicing as described under “Materials and Methods.” U2OS cells as described in the legend to Fig. 4 (G) and 293 cells as described in D. (H) were treated with tunicamycin for 1 or 3 h, pulsed with [35S]methionine prior to collecting protein, and run on a gel; total counts incorporated (top) were normalized to the amount of protein loaded (bottom), which was then quantitated for three biological replicates; S.E. values are shown. *, significant difference (p < 0.05 by Student's t test) from control time points. I, SK-N-(BE)2 cells, which have an inactive UPR and express predominantly eIF2Bδ V1, were engineered to overexpress eIF2Bδ V2. J, SK-N-(BE)2 cells expressing eIF2Bδ V2 were treated with tunicamycin for the times indicated, and ATF-4 induction was assessed by immunoblot. K, SK-N-(BE)2 cells expressing eIF2Bδ V2 were treated with tunicamycin, and protein synthesis was assessed as in G and H.

Article Snippet: Interactions of eIF2α and eIF2β were assessed with HA-tagged eIF2β and non-tagged eIF2α (provided by H. Harding), and interactions with eIF2Bβ and eIF2Bδ were done by first immunoprecipitating endogenous eIF2Bβ with antibody (H300, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) attached to fast flow protein A-Sepharose beads (Amersham Biosciences) and then immunoblotting with eIF2Bδ antibody.

Techniques: Expressing, Isolation, Western Blot, Activation Assay, Quantitation Assay, Real-time Polymerase Chain Reaction, Generated

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Unfolded Protein Response by eIF2B? Isoforms

doi: 10.1074/jbc.M110.153148

Figure Lengend Snippet: Expression of eIF2Bδ isoforms affects eIF2 and eIF2B interactions. A, an HA-tagged eIF2β plasmid was constructed, which interacts with eIF2α, as demonstrated by immunoprecipitation (IP) experiments. B, 293T cells were transfected with FLAG-tagged eIF2Bδ isoforms, and immunoprecipitation of endogenous eIF2Bβ was performed to determine the integrity of the eIF2B complex. C, eIF2α phosphorylation in 293T cells was assessed after treatment with tunicamycin. D, 293T cells were transfected with eIF2β and either control (pCDNA), V2 eIF2Bδ, or V1 eIF2Bδ. 24 h later, cells were treated with tunicamycin or mock-treated. After 3 h, cell lysates were incubated with anti-FLAG antibody to immunoprecipitate eIF2Bδ and immunoblotted for HA expression for eIF2β. A representative experiment is displayed; the experiment was repeated four times, and the average results with S.E. values (error bars) are shown.

Article Snippet: Interactions of eIF2α and eIF2β were assessed with HA-tagged eIF2β and non-tagged eIF2α (provided by H. Harding), and interactions with eIF2Bβ and eIF2Bδ were done by first immunoprecipitating endogenous eIF2Bβ with antibody (H300, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) attached to fast flow protein A-Sepharose beads (Amersham Biosciences) and then immunoblotting with eIF2Bδ antibody.

Techniques: Expressing, Plasmid Preparation, Construct, Immunoprecipitation, Transfection, Incubation