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1.
Figure 3

Figure 3. The E3 Interaction Network for UBA-UBX Proteins. From: UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

(A) The number and type of GG signature peptides identified by mass spectrometry in Flag–(UBA-UBX) immunoprecipitates is indicated. D – DMSO; M – MG132 (B) Osprey diagram illustrating the set of E3 ligases interacting with each UBA-UBX protein. (C) Quantitative representation of the interactions shown in B, obtained by calculating for each E3 ligase the abundance factor (AF) relative to p97. For details see Suppl. Table 2 and its legend.

Gabriela Alexandru, et al. Cell. ;134(5):804-816.
2.
Figure 1

Figure 1. Mammalian UBX-Domain Proteins Interact with p97 and Some Serve as Ubiquitin Receptors. From: UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

(A) The domain composition of human UBX proteins. Those identified in p97-Myc immunoprecipitates are indicated in red. UBA – Ubiquitin-associated; UIM – Ubiquitin-interacting motif; UBL – Ubiquitin-like; ThF – Thioredoxin-like fold. Further information about the respective domains can be found at http://www.ebi.ac.uk/interpro/. (B–D) N-terminally Flag-tagged UBX proteins were expressed in 293 cells and immunoprecipitated using anti-Flag beads. Cells expressing Flag-NPL4 or no Flag-tagged protein were used as positive and negative control, respectively. Some of the endogenous proteins that were coimmunoprecipitated are shown in western blots using specific antibodies. (Ubi)n refers to ubiquitin chains of varying length.

Gabriela Alexandru, et al. Cell. ;134(5):804-816.
3.
Figure 4

Figure 4. UBXD7 Interacts with Endogenous HIF1α Independently of p97. From: UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

(A) HIF1α peptides identified by mass spectrometry in Flag-UBXD7 immunoprecipitates from cells treated with MG132 for 2 h.
(B) The Flag–(UBA-UBX) immunoprecipitates shown in figure 2A were separated by PAGE and blotted using HIF1α specific antibodies (top). The bottom panel shows equivalent HIF1α levels in the input cell extracts.
(C) The specificity of HIF1α antibodies was tested on total cell extracts from HeLa cells treated with the indicated concentration of HIF1α siRNA, in the presence and in the absence of a 2 h treatment with MG132. A cross-reacting band partially overlapping with full-length HIF1α is indicated with *.
(D) Flag-UBXD7 was immunoprecipitated from HeLa cells treated with 5 nM of the indicated siRNAs for 48 h. Where indicated, 20 µM MG132 was added for 2 h prior to harvesting the cells. The indicated proteins were detected using specific antibodies, in the immunoprecipitates (left) and in the input cell extracts (right). Luc – luciferase

Gabriela Alexandru, et al. Cell. ;134(5):804-816.
4.
Figure 2

Figure 2. UBA-UBX Proteins Interact with Ubiquitinated Proteins Destined for Degradation and with Various E3 Ligases. From: UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

(A, B) Flag–(UBA-UBX) proteins were immunoprecipitated from 293 cells treated for 2 h with DMSO or MG132. Cells expressing no Flag-tagged protein were used as negative control. Some of the endogenous proteins that coimmunoprecipitated were detected by western blotting using specific antibodies. CUL2 input levels are shown at the bottom of panel B.
(C) The indicated Flag-tagged proteins were immunoprecipitated from HeLa cells treated with MG132 as above. AMSH, a protein that is not part of the p97 network, was used as negative control. UBXD7Δ and p47Δ are truncation mutants lacking the UBX domain. The UBA domain by itself was expressed at very low levels. The indicated proteins were detected using specific antibodies, in the immunoprecipitates (top) and in the input cell extracts (bottom). Ubi – ubiquitin

Gabriela Alexandru, et al. Cell. ;134(5):804-816.
5.
Figure 5

Figure 5. UBXD7 Recruits p97 to HIF1α. From: UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

(A) p97-Myc was immunoprecipitated from HeLa cells treated or not with 20 µM MG132 for 2 h prior to harvesting the cells. Cells expressing no Myc-tagged protein were used as negative control. The indicated proteins were detected in the immunoprecipitates (top) and in the input cell extracts (bottom) using specific antibodies.
(B) p97-Myc was immunoprecipitated from HeLa cells treated for 48 h with 5 nM of the indicated siRNAs and incubated with MG132 as above. The indicated proteins were detected using specific antibodies, in the immunoprecipitates (left) and in the input cell extracts (right). Luc – luciferase
(C) HeLa cell extracts were fractionated on a Superdex 200 gel filtration column. Individual fractions were concentrated by TCA precipitation and subjected to western blotting using specific antibodies. All proteins were endogenously expressed.

Gabriela Alexandru, et al. Cell. ;134(5):804-816.
6.
Figure 6

Figure 6. p97 Promotes HIF1α Degradation. From: UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

(A) Total cell extracts were prepared from cells treated with 5 nM of the indicated siRNA pools unless other siRNA concentration is specified. The siRNA treatment was 48 h and it was combined or not with 2 h of MG132 treatment. The indicated proteins were detected using specific antibodies. U7 – UBXD7
(B) HIF1α mRNA was amplified by RT-PCR using specific primers. 18S rRNA was amplified as control.
(C) Total cell extracts were prepared from cells treated with 5 nM of the indicated siRNA oligonucleotides or pools (p) for 48 h and incubated with 20 µM MG132 for 2 h. The indicated proteins were detected using specific antibodies.
A non-specific band cross-reacting with the HIF1α antibodies is indicated with *.
(D) UBXD7 Recruits p97/NPL4/UFD1 to the Ubiquitinated Substrate and Prevents Its Interaction with Other Proteasome Targeting Factors.
Top: 1 - UBA and UBX domains inactivate each other when the protein is not bound to the substrate. 2 - Substrate oligo-ubiquitination or attachment of multiple monoubiquitin allows recruitment of several UBA-UBX molecules per substrate. UBA domains mask the emerging ubiquitin-chain and prevent substrate degradation. 3 - Substrate binding frees the UBX domains that become available to recruit p97/NPL4/UFD1. The ubiquitin chain is elongated and the substrate is delivered to the proteasome for degradation.
Bottom: In the absence of UBXD7, other proteasome targeting factors mediate accelerated substrate degradation. The Rpn10/PSMD4 subunit of the proteasome is depicted as an alternative ubiquitin receptor. S – substrate, E3 – ubiquitin ligase

Gabriela Alexandru, et al. Cell. ;134(5):804-816.

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