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Results: 9

1.
FIG. 2

FIG. 2. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

p97/VCP/Cdc48p and PLAP are associated with mHDAC6. (A) Mouse testis cytoplasmic extracts were used to immunoprecipitate mHDAC6 with an anti-mHDAC6 antibody (lane −) or the same antibody preincubated with its target peptide (lane +). Bound proteins were eluted and analyzed by SDS-PAGE and revealed after silver staining. The indicated bands were excised and identified by mass spectrometry (MALDI-TOF analysis and MS/MS). (B) The identity of mHDAC6 in the immunoprecipitates was confirmed by Western blotting.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
2.
FIG. 3

FIG. 3. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

Homology between the mHDAC6 C-terminal domain and Ub-specific proteases (USPs). The sequence of the C-terminal region of mHDAC6 was aligned with that of several USPs from different species. Identical amino acids are boxed. The USP sequences are as follows: 1, human USP3, AF073344; 2, human USP20, AB023220; 3, Schizosaccharomyces pombe USP, AL021838; 4, Saccharomyces cerevisiae USP, P38237. Asterisks indicate histidines replaced by alanines described in a further experiment.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
3.
FIG. 5

FIG. 5. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

ZnF-UBP is the Ub-binding site of mHDAC6. (A) The indicated regions of mHDAC6 were cloned in an expression vector and used to generate 35S-labeled proteins in vitro. The pull-down was performed with Ub-agarose. (B) 35S-labeled mHDAC6 (full length or the C-terminal domain as indicated) bearing mutations in the ZnF-UBP (in m1, histidine 1098 is replaced by alanine, and in m2, histidines 1094 and 1098 are replaced [Fig. 3]) was produced and used in a Ub-agarose pull-down assay as described above. (C) mHDAC1, mHDAC4, and mHDAC5 were labeled in vitro and used in Ub pull-down assays as described above.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
4.
FIG. 6

FIG. 6. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

Identification of the major Ub-binding proteins present in mouse testis cytosolic extracts. (A) Proteins in mouse cytosolic testis extract retained on Ub-agarose beads were visualized on silver-stained SDS gel and compared with proteins retained on Ub-agarose beads when the pull-down was performed in the presence of an excess of free Ub (lane +). (B) The indicated bands were excised from a Coomassie blue-stained preparative SDS gel and identified by mass spectrometry as in Fig. 2. The table shows the identity of the excised bands. The right column shows the accession number and the corresponding database.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
5.
FIG. 1

FIG. 1. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

mHDAC6 is overexpressed in mouse testis. (A) Extracts prepared from the indicated mouse tissues were used to obtain a Western blot, which was successively probed with anti-HDAC6 and anti-HDAC1 antibodies. Fractions correspond to extracts prepared from spermatogenic cell populations enriched in the indicated cell types. P, pachytene; RS, round spermatids; RES, elongated and round spermatids. (B) Extracts from adult mouse testis (Testis) or from testis isolated from 6-day-old mice enriched in spermatogonia (Gonia), as well as cytoplasmic (C) and nuclear (N) extracts isolated from pachytene-enriched cells (P) were analyzed as in panel A. (C) A cryosection from adult mouse testis was used to immunodetect in situ mHDAC6 by using the anti-HDAC6 antibody (HDAC6 panel). The DNA panel shows corresponding Hoechst-labeled nuclei.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
6.
FIG. 9

FIG. 9. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

Ub recruits an active mHDAC6. Twenty microliters of immunopurified mHDAC6 (obtained as in Fig. 2) was incubated with 3H-labeled acetylated histones in the absence (lane 2) or in the presence of free Ub (lane 3). Lane 6 shows the inhibition of the deacetylase activity of the Ub-bound mHDAC6 by 100 ng of TSA per ml. The reaction mixtures containing or not containing free Ub were incubated with Ub-agarose and centrifuged, and labeled histones were added to the supernatant (Ub-agarose SN) to measure the deacetylase activity (lanes 4 and 5, respectively). Lane 1 shows the input of labeled histones. (Lower panels) As controls, the same experiments as those described above were performed, except that eluates from peptide-blocked anti-mHDAC6, obtained as described in the legend to Fig. 2, were analyzed.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
7.
FIG. 4

FIG. 4. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

mHDAC6 specifically interacts with Ub. (A) Mouse testis cytoplasmic extracts were incubated with an Ub-agarose matrix (lane 1). In control experiments, the indicated amounts of free Ub (lanes 2 to 4) or BSA (lanes 5 to 7) were added to the extract before the addition of the Ub-agarose. After the pull-down, bound proteins were eluted and analyzed by Western blotting with an anti-mHDAC6 antibody. (B and C) Ten microliters of supernatant after the pull-down (B) and the same amounts of the input materials (C) were used to monitor the presence of mHDAC6. (D) mHDAC6 interacts with Ub-fusion proteins, but not with the GST–SUMO-1 fusion. The pull-down was performed as described above with the immobilized indicated GST-fusion proteins. Bound proteins were analyzed as described above with an anti-mHDAC6 antibody. H4Nt, the N-terminal H4 histone tail.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
8.
FIG. 8

FIG. 8. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

Ub binding by mHDAC6 induces the release of p97/VCP/Cdc48p and PLAP. mHDAC6 from mouse cytosolic extracts was immunoprecipitated (IP)as described in the legend to Fig. 2 in the presence or absence of free Ub (+ and − lanes, respectively). Immunoprecipitated proteins were eluted with the immunogenic peptide and analyzed on a silver-stained gel. (This experiment was performed in parallel with that shown in Fig. 2A, and Fig. 2A and 8A are from the same gel.) The positions of mHDAC6, p97/VCP/Cdc48p, and PLAP are indicated. (B) Prior to immunoprecipitations of mHDAC6, the mouse testis extracts were preincubated with or without ATP (2 mM) and/or Ub as indicated. The presence of mHDAC6 and p97/VCP/Cdc48p in the immunoprecipitates was then determined by Western blotting with the corresponding antibodies. The input panel represents 2% of the amount of extract used in the immunoprecipitation. (C) Mouse cytosolic extracts were subjected to Ub pull-down with Ub-agarose beads as described in the legends to Fig. 4 and 6. Unbound materials present in the supernatant (SN) after the pull-down and bound materials retained on Ub-agarose beads (P) were used to monitor the presence of p97/VCP/Cdc48p and mHDAC6 with the corresponding antibodies.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.
9.
FIG. 7

FIG. 7. From: Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways.

Ub binding by mHDAC6 C-terminal region interferes with in vitro ubiquitination of proteins present in mouse testis cytosolic extract. (A) mHDAC6 binds directly to Ub. mHDAC6 C-terminal end (amino acids 824 to 1149) was cloned in an expression vector (pET-28a+; NOVAGEN). His-HDAC6 C-terminal region was produced and purified and used to perform Ub-agarose pull-down experiments in the absence (−) or presence (+) of free Ub or BSA as indicated. After the pull-down, bound proteins were eluted and analyzed by Western blotting with an anti-mHDAC6 antibody. I, indicates 50% of the input material; lanes labeled Pull down show materials eluted after Ub-agarose pull-down. (B) Mouse testis cytosolic extract is capable of directing an in vitro ubiquitation of extract proteins in an ATP-dependent manner. Cytosolic testis extracts were incubated in the absence (−) or presence (+) of ATP and increasing amounts of the purified mHDAC6 C-terminal fragment (lanes 3 and 4) and the same number of molecules of BSA (lanes 5 and 6). After incubation, a fraction of the extract was used to monitor protein ubiquitination with a Western blot and an anti-Ub antibody (Santa Cruz). The right panel shows that the addition of free Ub relieves the mHDAC6 C-terminal-mediated repression of protein ubiquitination (compare lanes 9 and 10). (C) The C-terminal region of mHDAC6 (the same fragment described above) from the wild-type protein or the m2 mutants (Fig. 3 and 5B) was fused to GST, expressed in bacteria, and purified (GST-Cter WT and GST-Cter m2, respectively). Increasing amounts of recombinant proteins were added to the extract as described above, and protein ubiquitination was monitored (upper panel). The lower panel shows the amount of GST-fusion proteins added to the extract in this experiment by analyzing a fraction of the samples with an anti-GST antibody. Lane 1 shows a standard ubiquitination reaction, and lane 8 shows the same reaction without ATP.

Daphné Seigneurin-Berny, et al. Mol Cell Biol. 2001 December;21(23):8035-8044.

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