UBQLN1 (D3T7F) Rabbit mAb 14526S from Cell Signaling Technology

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The Journal of biological chemistry
May 01 2022

Fig 1: Inhibiting the retroviral protease site of Ddi2 enhances the binding of Ddi2 to Ub conjugates but decreases binding to proteasomes.A, the design of the experiment in (B) to test the effects of the Ddi2 protease activity on its association with Ub conjugates. Lysates of HAP1 Ddi2 knockout cells were treated with 1 µM bortezomib (BTZ) for 4 h (to obtain a high content of Ub conjugates), but no Ddi2 was mixed with lysates of HAP1 Ddi2 knockout cells transfected with WT Ddi2 or the inactive mutant D252V Q256L Ddi2 (DV/QL) and treated with 10 µM of the E1 inhibitor TAK-243 (Eli) for 4 h (to obtain high levels of WT or inactive Ddi2 and no Ub conjugates). After these lysates were combined, the lysates were incubated at 37 °C for 1 h, and the changes in levels of the Ub conjugates were analyzed at different times. B, inactive Ddi2 protects Ub conjugates from deubiquitylation. Lysates containing ubiquitylated proteins but no Ddi2 were mixed with lysates containing high levels of Ddi2, but lacking Ub conjugates as described in (A). The mixed lysates (left three lanes) were incubated at 37 °C for 1 h with 1.6 µM Ub aldehyde (Ub-Al) to block DUb activity and 10 µM E1i to prevent further ubiquitylation, or 10 µM BTZ, which allows DUb function but blocks proteasomal degradation. C, inactive Ddi2 protects ubiquitylated E6AP from deubiquitylation. HAP1 Ddi2 knockout cells were transfected with WT or D252V Q256L Ddi2 and treated with 10 µM of the E1 inhibitor Eli for 4 h as in (B). Ubiquitylated GST-E6AP was added to the lysates and incubated at 37 °C for up to 3 h. D, inhibition of the proteolytic activity of Ddi2 with nelfinavir (10 µM, 30 min) increases the binding of Ddi2 and UBQLN1 but not Rad23B to ubiquitylated E6AP. Lysates of HEK293-HBTH-Rpn1 were incubated with ubiquitylated GST-E6AP on immobilized glutathione-Sepharose for 1 h at 4 °C, and immunoblots for different proteins were quantitated. E, mutation of the Ddi2 protease site increases the binding of Ddi2 to ubiquitylated E6AP. Lysates of WT Ddi2 and D252N mutant HCT116 cells were incubated as in (D) with ubiquitylated E6AP. Quantification of immunoblots of Ddi2 that bound to ubiquitylated GST-E6AP as the percent binding of proteins from mutant cells versus the WT. F, inhibition of the Ddi2 protease site with nelfinavir decreases the binding of Ddi2 to 26S proteasomes. HEK293-HBTH-Rpn11 cells were treated with 10 µM nelfinavir or with vehicle (methanol) for 30 min, and lysates were incubated with streptavidin resin to bind 26S proteasomes. The amount of Ddi2 present was then assayed. (n = 4; mean; *p < 0.05, **p < 0.01, ns). DUb, deubiquitylating enzyme; GST, glutathione-S-transferase; HEK293, human embryonic kidney 293 cell line; ns, not significant; Ub, ubiquitin.
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Neoplasia
March 01 2022

Fig 2: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 cells following RNAseq analysis. (B) Representative list of top 10 genes down-regulated specifically by siUBQLN1. (C) Representative list of top 10 genes down-regulated specifically by siUBQLN2. (D) Representative list of top 10 genes up-regulated specifically by siUBQLN1. (E) Representative list of top 10 genes up-regulated specifically by siUBQLN2. (F) Representative bar graph showing confirmation of genes that were significantly altered following loss of UBQLN1 in A549 and HOP62 cells. (G) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in SK-N-BE(2) cells following RNAseq analysis. (H) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells following RNAseq analysis.
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Neoplasia
March 01 2022

Fig 3: shUbqln1 mES cells Differentiation into Neuronal or Epithelial-like cells. Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. (A) Schematic of doxycycline inducible UBQLN1 shRNA differentiation into mouse embryonic stem cell neuronal (mESN) and mouse embryonic stem cell epithelial (mESE) cells. (B) Representative microscopy images of unmanipulated mES cells and mES cells following doxycycline induction. (C) Representative t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of unmanipulated neuronal/differentiated neuronal and unmanipulated epithelial/differentiated epithelial cells. Neuronal cells revealed identical transcriptional profile throughout the cell populations (green vs black spots) while loss of UBQLN1 found to alter differentiation of epithelial cells (blue vs red spots). (D) Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. UBQLNs has N-terminal UBL domain (ubiquitin-like), C-terminal UBA domain (ubiquitin-associated) and STI regions in the middle. Ubiquilin2 has an additional collagen-like domain between the fourth STI and UBA domains.
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Neoplasia
March 01 2022

Fig 4: UBQLN1 and UBQLN2 interacts with each other. (A) Representative chart showing number of unique peptides identified each time following IP MS/MS. 293T cells were transfected with FLAG-UBQLN1 (PCS2-PLIC1). Immunoprecipitation was performed by using flag conjugated beads to pull down UBQLN1 and endogenous UBQLN2 followed by mass spectrometry analysis. Experiment was performed four separate times and each time unique peptides were identified following IP MS/MS were represented in chart. (B) Western blot analysis of UBQLN1 interaction with UBQLN2. 293T cells were transfected either with empty vector (EV) or with FLAG-UBQLN1 (PCS2-PLIC1) and FLAG-UBQLN1 (PCS2-PLIC1-CL) following crosslinking with formalin by heating protein lysates in SDS at indicated temperatures. 72 h post transfection cells were lysed, and western blots were performed with the indicated antibody. Where indicated immunoprecipitation with anti-FLAG conjugated agarose beads was performed to determine UBQLN1 interacting complexes.
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Neoplasia
March 01 2022

Fig 5: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in differentiated iPSC-WNT and mESC-EGFR cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in unmanipulated iPSC and differentiated iPSC-WNT epithelial and iPSC-EGF neuronal cells following RNAseq analysis. (B) Representative list of overlapping 17 genes that were significantly altered following UBQLN1 knockdown in unmanipulated iPSC, differentiated iPSC-WNT epithelial and iPSC-EGF neuronal cells following RNAseq analysis. (* = found in A549 UBQLN1/UBQLN2 knockdown common signature). (C) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 knockdown in unmanipulated mESC, differentiated mESC-WNT epithelial and mESC-EGF neuronal cells following RNAseq analysis. (D) Representative list of overlapping 21 genes that were significantly altered following UBQLN1 knockdown in unmanipulated mESC, differentiated mESC-WNT epithelial and mESC-EGF neuronal cells following RNAseq analysis.

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UBQLN1 (D3T7F) Rabbit mAb

The UBQLN1 (D3T7F) Rabbit mAb Antibody from Cell Signaling Technology is a Rabbit Monoclonal antibody to Ubiquilin 1, and UBQLN1. This antibody recognizes Homo Sapiens, Human, and Non-Human Primate antigen. The UBQLN1 (D3T7F) Rabbit mAb Antibody has been shown to work in the following applications: Precipitation, Immunoprecipitation, and Western Blot.

Description

UBQLN1 (D3T7F) Rabbit mAb recognizes endogenous levels of total UBQLN1 protein. This antibody does not cross-react with other UBQLN proteins

The Journal of biological chemistry

Neoplasia

Neoplasia

Neoplasia

Neoplasia

UBQLN1 (D3T7F) Rabbit mAb from Cell Signaling Technology

Description

Cell Signaling Technology

Citations (12)

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UBQLN1 (D3T7F) Rabbit mAb from Cell Signaling Technology

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