Journal:

Article Title: Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

doi:

Figure Lengend Snippet: Activation of HSF2 by treatment of cells with specific proteasome inhibitors. (A) Gel mobility shift assays to analyze formation of HSF-HSE complexes using whole-cell extracts prepared from untreated K562, HeLaS3, and HepG2 cells and MEF (lanes 1, 5, 8, and 10, respectively), from K562 cells treated with hemin for 12 h (lane 2) or with MG132 or lactacystin (Lac) for 2 h (lanes 3 and 4, respectively), from HeLaS3 cells treated with MG132 or lactacystin for 6 h (lanes 6 and 7, respectively), from HepG2 cells treated with MG132 for 2 h (lane 9), and from MEF treated with MG132 for 2 h (lane 11). (B) Identification of the DNA-binding activity as primarily HSF2 by antibody supershift assays. Extracts from hemin- and MG132-treated K562 cells (lanes 1 to 3 and 4 to 6, respectively) were incubated either with or without a 1:50 dilution of specific HSF2 or HSF1 antisera, as indicated, prior to the gel mobility shift assay. Similarly, extracts from MEF heat shocked at 42°C for 1 h (lanes 7 to 9) or treated with MG132 for 6 h (lanes 10 to 13) were incubated in the presence or absence of either the antiserum specific to HSF1, the antiserum specific to HSF2, or both. The HSF DNA-binding activities are indicated by arrows. NS, nonspecific binding.

Article Snippet: The human tissue culture cell lines K562 (grown in RPMI 1640 supplemented with 10% fetal calf serum), HeLaS3 (grown in Joklik’s medium with 5% calf serum), and HepG2 (grown in Eagle’s minimal essential medium with 10% fetal calf serum, sodium pyruvate, and nonessential amino acids), and mouse embryo fibroblasts (MEF; a gift of I. J. Benjamin, Southwestern Medical School) (grown in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum, nonessential amino acids, and 0.5 μM β-mercaptoethanol), were treated with 20 μM bovine hemin (Aldrich), 10 μM cycloheximide (Sigma), 10 μM MG132 (Peptides International), or 10 μM lactacystin (E. J. Corey, Harvard University) as indicated.

Techniques: Activation Assay, Mobility Shift, Binding Assay, Activity Assay, Incubation

Journal:

Article Title: Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

doi:

Figure Lengend Snippet: Coordinate changes in HSF2 DNA-binding activity and protein levels, determined by gel mobility shift (upper panels) and immunoblot (lower panels) assays of whole-cell extracts from MEF treated with MG132 for up to 6 h (A), ts85 cells incubated at the nonpermissive temperature for up to 4 h in the presence (lanes 4 and 5) or absence (lanes 2 and 3) of cycloheximide (CHX) (B), K562 cells left untreated (lane 1) or treated for 2 h with MG132 alone (lane 2) or with MG132 and cycloheximide (lane 3) (C), and K562 cells left untreated (lane 1) or treated with MG132 for 6 h and allowed to recover for 0 (lane 2), 4 (lane 3), and 10 (lane 4) h in inhibitor-free medium (D). C, control.

Article Snippet: The human tissue culture cell lines K562 (grown in RPMI 1640 supplemented with 10% fetal calf serum), HeLaS3 (grown in Joklik’s medium with 5% calf serum), and HepG2 (grown in Eagle’s minimal essential medium with 10% fetal calf serum, sodium pyruvate, and nonessential amino acids), and mouse embryo fibroblasts (MEF; a gift of I. J. Benjamin, Southwestern Medical School) (grown in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum, nonessential amino acids, and 0.5 μM β-mercaptoethanol), were treated with 20 μM bovine hemin (Aldrich), 10 μM cycloheximide (Sigma), 10 μM MG132 (Peptides International), or 10 μM lactacystin (E. J. Corey, Harvard University) as indicated.

Techniques: Binding Assay, Activity Assay, Mobility Shift, Western Blot, Incubation

Journal:

Article Title: Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

doi:

Figure Lengend Snippet: Inhibition of protein synthesis results in a loss of HSF2 DNA-binding activity and of HSF2 protein levels. (A) Gel mobility shift (upper panel) and immunoblot (lower panel) assays of whole-cell extracts from K562 cells left untreated (lane 1) or induced with hemin for 12 h and treated with cycloheximide (CHX) for 0 (lane 2), 30 (lane 3), or 120 (lane 4) min. (B) The effects of simultaneous inclusion of cycloheximide and MG132, for 0 (lane 1), 30 (lane 2), and 120 (lane 3) min, on hemin-induced cells were also assessed. (C) Glycerol gradient fractionation (55) of K562 cell extracts from control cells (I) and from cells induced with hemin for 12 h (II to IV) and treated with cycloheximide for 0 (II), 30 (III), or 120 (IV) min. Fractions were collected from the top to the bottom of the gradients (fractions 2 to 16). The positions corresponding to dimeric and trimeric HSF2 are shown. The S values from protein standards are indicated (cytochrome c, 1.9S; bovine serum albumin, 4.3S; alcohol dehydrogenase, 7.4S).

Article Snippet: The human tissue culture cell lines K562 (grown in RPMI 1640 supplemented with 10% fetal calf serum), HeLaS3 (grown in Joklik’s medium with 5% calf serum), and HepG2 (grown in Eagle’s minimal essential medium with 10% fetal calf serum, sodium pyruvate, and nonessential amino acids), and mouse embryo fibroblasts (MEF; a gift of I. J. Benjamin, Southwestern Medical School) (grown in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum, nonessential amino acids, and 0.5 μM β-mercaptoethanol), were treated with 20 μM bovine hemin (Aldrich), 10 μM cycloheximide (Sigma), 10 μM MG132 (Peptides International), or 10 μM lactacystin (E. J. Corey, Harvard University) as indicated.

Techniques: Inhibition, Binding Assay, Activity Assay, Mobility Shift, Western Blot, Fractionation

Journal:

Article Title: Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

doi:

Figure Lengend Snippet: Elevated synthesis and decreased degradation of HSF2 upon proteasome inhibition. (A) Control MEF (lane 1) and MEF treated with MG132 for 2 (lane 2) and 6 (lane 3) h were pulse-labeled for 15 min, following which cell extracts were prepared for use for immunoprecipitation (upper panel) and immunoblot (lower panel) analyses as described above. The labeled proteins were visualized by fluorography and quantitated by PhosphorImager analysis. The labeled protein band appearing above 208 kDa represents a nonspecific interaction with the antibodies. (B) MEF treated with MG132 for 4 h were pulse-labeled for 15 min and incubated in complete medium with (lane 3) or without (lane 2) MG132 for an additional 4 h. Cell extracts were prepared and used for immunoprecipitation analyses as described above.

Article Snippet: The human tissue culture cell lines K562 (grown in RPMI 1640 supplemented with 10% fetal calf serum), HeLaS3 (grown in Joklik’s medium with 5% calf serum), and HepG2 (grown in Eagle’s minimal essential medium with 10% fetal calf serum, sodium pyruvate, and nonessential amino acids), and mouse embryo fibroblasts (MEF; a gift of I. J. Benjamin, Southwestern Medical School) (grown in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum, nonessential amino acids, and 0.5 μM β-mercaptoethanol), were treated with 20 μM bovine hemin (Aldrich), 10 μM cycloheximide (Sigma), 10 μM MG132 (Peptides International), or 10 μM lactacystin (E. J. Corey, Harvard University) as indicated.

Techniques: Inhibition, Labeling, Immunoprecipitation, Western Blot, Incubation

Journal:

Article Title: Heat Shock Response and Protein Degradation: Regulation of HSF2 by the Ubiquitin-Proteasome Pathway

doi:

Figure Lengend Snippet: Induction of heat shock proteins in K562 cells by proteasome inhibitor treatment. (A) 35S-labeled cell extracts from pulse-labeled cells analyzed by SDS–10% PAGE. Cells were left untreated (lane 1), treated with 10 μM MG132 for 2 or 6 h (lanes 2 and 3), treated with 10 μM lactacystin (Lac) for 2 or 6 h (lanes 4 and 5), or heat shocked (HS) at 42°C for 1 h (lane 6). (B) Hsp70 immunoblot of cell extracts using the mouse monoclonal antibody 4G4. From left to right, treatments correspond to those described for panel A, lanes 1 through 6. (C) Hdj-1 immunoblot of cell extracts using rabbit polyclonal sera raised against Hdj-1. From left to right, treatments correspond to those described for panel A, lanes 1 through 6. (D through F) Two-dimensional protein gel analysis of 35S-labeled cell extracts left untreated (D), treated with 10 μM MG132 for 6 h (E), or heat shocked at 42°C for 1 h (F). The first dimension was isoelectric focusing generating a gradient from pH 3 to 10. The second dimension was SDS–10% PAGE. Open arrowhead, location of Hsc70; solid arrowhead, location of Hsp70; arrow, location of Hsp90. The circled proteins of approximately 35 kDa are induced preferentially upon MG132 treatment. The protein spot labeled a corresponds to actin.

Article Snippet: The human tissue culture cell lines K562 (grown in RPMI 1640 supplemented with 10% fetal calf serum), HeLaS3 (grown in Joklik’s medium with 5% calf serum), and HepG2 (grown in Eagle’s minimal essential medium with 10% fetal calf serum, sodium pyruvate, and nonessential amino acids), and mouse embryo fibroblasts (MEF; a gift of I. J. Benjamin, Southwestern Medical School) (grown in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum, nonessential amino acids, and 0.5 μM β-mercaptoethanol), were treated with 20 μM bovine hemin (Aldrich), 10 μM cycloheximide (Sigma), 10 μM MG132 (Peptides International), or 10 μM lactacystin (E. J. Corey, Harvard University) as indicated.

Techniques: Labeling, Western Blot