Journal: International Journal of Molecular Sciences

Article Title: A Novel Mechanism of Immunoproteasome Regulation via miR-369-3p in Intestinal Inflammatory Response

doi: 10.3390/ijms232213771

Figure Lengend Snippet: PA28α and PA28β protein expression modulated by miR-369-3p in RAW264.7 cells. Western blot analysis of PA28α ( A ) and PA28β ( B ) proteins after miR-369-3p mimic transfection in the RAW264.7 cell line, both unstimulated and LPS-stimulated. In basal conditions, miR-369-3p transfection did not influence the expression of these proteins. In LPS-stimulated cells, the miR-369-3p transfection resulted in a significant reduction in PA28α and PA28β. Vinculin was used as housekeeping protein to normalize the data. Data were obtained by comparing normalized sample values to the normalized mock sample value (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary antibodies used were rabbit pAb PSMB9 (Invitrogen, #PA1-1960), rabbit mAb PSMB8/LMP7 (Cell Signaling, Technology, Danvers, MA, USA, #13635), rabbit mAb Pa28α (Cell Signaling, Technology, Danvers, MA, USA, #9643), rabbit mAb Pa28β (Cell Signaling, Technology, Danvers, MA, USA, #2409), and rabbit mAb Vinculin (Cell Signaling, Technology, Danvers, MA, USA, #13901).

Techniques: Expressing, Western Blot, Transfection

Journal: Cancers

Article Title: Association of Proteasome Activity and Pool Heterogeneity with Markers Determining the Molecular Subtypes of Breast Cancer

doi: 10.3390/cancers17010159

Figure Lengend Snippet: Proteasome activities and content in BC and adjacent tissues. Representative immunohistochemical staining of tumor tissues ( a ). First row ( A1 – A4 ) Invasive carcinoma of nonspecific type (Luminal A subtype): A1 —Strong expression of ER; A2 —Strong expression of PR; A3 —negative expression of HER-2; A4 —low level of Ki-67. Second row ( B1 – B4 ) Invasive carcinoma of nonspecific type (Luminal B subtype): B1 —Strong expression of ER; B2 —Strong expression of PR; B3 —weak (1+) expression of HER-2; A4 —high level of Ki-67 (>20%). Third row ( C1 – C4 ) Invasive carcinoma of nonspecific type (Triple negative subtype): C1 —negative expression of ER; C2 , negative expression of PR; C3 , negative expression of HER-2; A4 , high level of Ki-67 (>50%). Immunohistochemistry, ×200. Scale bar—100 μm. ( b ) ChTL and CL proteasome activities in BC and adjacent tissues ( c ). Proteasome activities in tissues of various molecular subtypes of BC. ( d ) The content of 20S proteasome or proteasome regulator subunits ((α1, α2, α3, α5, α6, α7), Rpt6, PA28β LMP-2, LMP-7) determined by Western blotting in BC (1) and adjacent tissues (2). Representative Western blots are shown. ( e ) Content of proteasome subunits in tumour and adjacent tissue; and in different molecular subtypes of the BC ( f ) determined via quantification of blots using the ImageJ software ver. 1.54i. Red box indicates the relative content of proteasome subunits in the adjacent tissue, which was considered as 100%; * p < 0.05, ** p < 0.01, Mann-Whitney test.

Article Snippet: Primary mouse monoclonal antibodies to 20S proteasome subunits α1, α2, α3, α5, α6, α7 (cat. # sc-58412), mouse mAbs to LMP-7 (cat. # sc-100284), rabbit polyclonal antibodies (pAbs) to LMP-2 (cat. # sc-28809), and goat antibodies to the subunit PA28β (cat. # sc-23642) were obtained from Santa Cruz Biotechnology, Dallas, TX, USA.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunohistochemistry, Western Blot, Software, MANN-WHITNEY

Journal: Cancers

Article Title: Association of Proteasome Activity and Pool Heterogeneity with Markers Determining the Molecular Subtypes of Breast Cancer

doi: 10.3390/cancers17010159

Figure Lengend Snippet: Relative content of proteasome subunits in tumor tissue in different molecular subtypes of BC.

Article Snippet: Primary mouse monoclonal antibodies to 20S proteasome subunits α1, α2, α3, α5, α6, α7 (cat. # sc-58412), mouse mAbs to LMP-7 (cat. # sc-100284), rabbit polyclonal antibodies (pAbs) to LMP-2 (cat. # sc-28809), and goat antibodies to the subunit PA28β (cat. # sc-23642) were obtained from Santa Cruz Biotechnology, Dallas, TX, USA.

Techniques:

Journal: PLoS ONE

Article Title: Immunoproteasome Overexpression Underlies the Pathogenesis of Thyroid Oncocytes and Primary Hypothyroidism: Studies in Humans and Mice

doi: 10.1371/journal.pone.0007857

Figure Lengend Snippet: Comparison of gene expression by SAGE in thyrocytes isolated from thyr -IFNγ transgenic (TG) or wild type (WT) mice (shows the genes that displayed the highest TG over WT ratio).

Article Snippet: After blocking aspecific binding with 5% normal goat serum, sections were incubated with the primary antibody; rabbit anti proteasome subunit (LMP2, LMP7, LMP10, PA28α, and PA28β; Biomol International, L.P., Plymouth Meeting, PA), in PBS supplemented with 1% bovine serum albumin, and incubated overnight at 4°C in a humid chamber.

Techniques: Comparison, Gene Expression, Isolation, Transgenic Assay, Membrane

Journal: PLoS ONE

Article Title: Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

doi: 10.1371/journal.pone.0067793

Figure Lengend Snippet: Four pairs of HS-Csn8KO and CTL littermate mice at 4-week-old were used for analyses presented here. ( A ) Representative images of western blot analyses of the indicated Bcl2 family proteins. β-Tubulin was probed as loading control (B~D). Summary of quantitative analyses. Pooled densitometry data of Bim ( B ), Bax ( C ), Bcl2 and Bcl-xL ( D ) normalized to β-tubulin are presented. *p<0.05, **p<0.01 vs. CTL; n=4 mice/group.

Article Snippet: Western blots were performed with following antibodies: Rpn2, Rpn8, Rpn10, Rpt5, PA28α, PA28β, PA28γ, VHL (von Hippel–Lindau), HIF1α (hypoxia inducible factor 1α), and Csn8 (Biomol), Bim and Bcl-xL (Chemicon), Bax and Bcl2 (BD Pharmingen Tech.), Nedd8 (Alexis), β-tubulin and ubiquitin (Sigma), CIS (cytokine- i nducible Src homology 2 domain-containing protein) and GFP (Santa Cruz Biotechnology), and 20S subunit β5 (custom made).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

doi: 10.1371/journal.pone.0067793

Figure Lengend Snippet: Crude proteins extracts from liver tissues were used for immunoprecipitation (IP) followed by iimunoblotting (IB) analyses. ( A ) Immunoprecipitation using a Bim-specific antibody followed by IB for the indicated proteins. ( B ) Pooled densitometry data of the IP western blot analyses as illustrated in panel A. For each protein, the density of the Csn8KO group is shown as the relative value to the average value of the corresponding CTL group. Arbitrary unit is used. ( C ) Immunoprecipitation of Bcl2 was followed by IB for Bcl2, Bim, and Bax. NS denotes a non-specific band. ( D ) Pooled densitometry data of the IP western blot analyses as illustrated in panel C. ( E ) Immunoprecipitation of Bax was followed by IB for Bax and Bim. *p<0.05, **p<0.01 vs. CTL.

Article Snippet: Western blots were performed with following antibodies: Rpn2, Rpn8, Rpn10, Rpt5, PA28α, PA28β, PA28γ, VHL (von Hippel–Lindau), HIF1α (hypoxia inducible factor 1α), and Csn8 (Biomol), Bim and Bcl-xL (Chemicon), Bax and Bcl2 (BD Pharmingen Tech.), Nedd8 (Alexis), β-tubulin and ubiquitin (Sigma), CIS (cytokine- i nducible Src homology 2 domain-containing protein) and GFP (Santa Cruz Biotechnology), and 20S subunit β5 (custom made).

Techniques: Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

doi: 10.1371/journal.pone.0067793

Figure Lengend Snippet: Four pairs of HS-Csn8KO and CTL littermate mice at 4-week-old were used for analyses presented here. ( A ) Representative images of western blot analyses of the indicated Bcl2 family proteins. β-Tubulin was probed as loading control (B~D). Summary of quantitative analyses. Pooled densitometry data of Bim ( B ), Bax ( C ), Bcl2 and Bcl-xL ( D ) normalized to β-tubulin are presented. *p<0.05, **p<0.01 vs. CTL; n=4 mice/group.

Article Snippet: Western blots were performed with following antibodies: Rpn2, Rpn8, Rpn10, Rpt5, PA28α, PA28β, PA28γ, VHL (von Hippel–Lindau), HIF1α (hypoxia inducible factor 1α), and Csn8 (Biomol), Bim and Bcl-xL (Chemicon), Bax and Bcl2 (BD Pharmingen Tech.), Nedd8 (Alexis), β-tubulin and ubiquitin (Sigma), CIS (cytokine- i nducible Src homology 2 domain-containing protein) and GFP (Santa Cruz Biotechnology), and 20S subunit β5 (custom made).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

doi: 10.1371/journal.pone.0067793

Figure Lengend Snippet: Crude proteins extracts from liver tissues were used for immunoprecipitation (IP) followed by iimunoblotting (IB) analyses. ( A ) Immunoprecipitation using a Bim-specific antibody followed by IB for the indicated proteins. ( B ) Pooled densitometry data of the IP western blot analyses as illustrated in panel A. For each protein, the density of the Csn8KO group is shown as the relative value to the average value of the corresponding CTL group. Arbitrary unit is used. ( C ) Immunoprecipitation of Bcl2 was followed by IB for Bcl2, Bim, and Bax. NS denotes a non-specific band. ( D ) Pooled densitometry data of the IP western blot analyses as illustrated in panel C. ( E ) Immunoprecipitation of Bax was followed by IB for Bax and Bim. *p<0.05, **p<0.01 vs. CTL.

Article Snippet: Western blots were performed with following antibodies: Rpn2, Rpn8, Rpn10, Rpt5, PA28α, PA28β, PA28γ, VHL (von Hippel–Lindau), HIF1α (hypoxia inducible factor 1α), and Csn8 (Biomol), Bim and Bcl-xL (Chemicon), Bax and Bcl2 (BD Pharmingen Tech.), Nedd8 (Alexis), β-tubulin and ubiquitin (Sigma), CIS (cytokine- i nducible Src homology 2 domain-containing protein) and GFP (Santa Cruz Biotechnology), and 20S subunit β5 (custom made).

Techniques: Immunoprecipitation, Western Blot

Journal: Frontiers in Oncology

Article Title: PA28α/β Promote Breast Cancer Cell Invasion and Metastasis via Down-Regulation of CDK15

doi: 10.3389/fonc.2019.01283

Figure Lengend Snippet: Knockdown of PA28α/β represses breast tumor cell migration and invasion. (A) RT-PCR assay was performed to analyze PA28α/β expression in breast cancer cell lines MDA-MB-231, MDA-MB-453, and BT549 that transfected with scramble control and siRNA (*** P < 0.001). (B) CCK8 assay was used to detect the growth rate of PA28α/β-knockdown cells and the scramble-siRNA-control transfected cells. (C) Transient knockdown of PA28α or PA28β in breast cancer cells reduced invasion as revealed by Transwell assay. Representative images and quantification data are shown (NS, P >0.05; * P < 0.05; ** P < 0.005; *** P < 0.001). (D) SiRNA-mediated double knockdown of PA28α/β inhibited cell invasion as detected by Transwell assay. Representative images and quantification data are shown (*** P < 0.001). # Indicates no significance.

Article Snippet: Primary antibodies specific for CDK15 (GeneTex 1:50), PA28α (Cell signaling 1:100), PA28β (Cell signaling 1:100) were used for immunohistochemistry.

Techniques: Knockdown, Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, CCK-8 Assay, Transwell Assay

Journal: Frontiers in Oncology

Article Title: PA28α/β Promote Breast Cancer Cell Invasion and Metastasis via Down-Regulation of CDK15

doi: 10.3389/fonc.2019.01283

Figure Lengend Snippet: PA28α/β proteins are responsible for breast cancer cell metastasis. (A) Stable silencing of PA28α/β by shRNA repressed invasion of MDA-MB-231 cells as revealed by Transwell assay. Representative images and quantification data are shown (*** P < 0.001). (B) Stable silencing of PA28α/β in MDA-MB-231 cells by shRNA suppressed migration which detected by wound healing assay. Representative images and quantification data are shown (** P < 0.005; *** P < 0.001). (C) Photographs of pulmonary metastasis nodules under macro-and microscope. Images of H&E staining were captured using 40× (middle) and 200× (bottom) magnitudes, respectively. Numbers of lung tumor nodules are shown (*** P < 0.001).

Article Snippet: Primary antibodies specific for CDK15 (GeneTex 1:50), PA28α (Cell signaling 1:100), PA28β (Cell signaling 1:100) were used for immunohistochemistry.

Techniques: shRNA, Transwell Assay, Migration, Wound Healing Assay, Microscopy, Staining

Journal: Frontiers in Oncology

Article Title: PA28α/β Promote Breast Cancer Cell Invasion and Metastasis via Down-Regulation of CDK15

doi: 10.3389/fonc.2019.01283

Figure Lengend Snippet: Inhibition of immunoproteasome rescues the protein expression of CDK15. (A) CDK15 protein levels were detected in three breast cancer cell lines when PA28α and/or PA28β was knocked down by siRNA. (B) CDK15 protein expression was observed in three breast cancer cell lines when β5i was stably silenced by shRNA. (C) Cell invasive ability was observed in vector control and β5i-knockdown breast cancer cells. Representative images and quantification data are shown (** P < 0.005; *** P < 0.001).

Article Snippet: Primary antibodies specific for CDK15 (GeneTex 1:50), PA28α (Cell signaling 1:100), PA28β (Cell signaling 1:100) were used for immunohistochemistry.

Techniques: Inhibition, Expressing, Stable Transfection, shRNA, Plasmid Preparation, Control, Knockdown

Journal: Frontiers in Oncology

Article Title: PA28α/β Promote Breast Cancer Cell Invasion and Metastasis via Down-Regulation of CDK15

doi: 10.3389/fonc.2019.01283

Figure Lengend Snippet: PA28α/β-induced cell migration and invasion partially depend on down-regulation of CDK15. (A) Breast cancer cells were singly transfected with siRNA of PA28α, PA28β and CDK15, or were co-silenced with siRNA of PA28α/CDK15 and PA28β/CDK15. Cell invasive ability was measured by Transwell assay and typical images are shown. (B) Breast cancer cells were singly transfected with siRNA of PA28α, PA28β and CDK15, or were co-silenced with siRNA of PA28α/CDK15 and PA28β/CDK15. Cell migration was observed by wound healing assay and typical images are shown.

Article Snippet: Primary antibodies specific for CDK15 (GeneTex 1:50), PA28α (Cell signaling 1:100), PA28β (Cell signaling 1:100) were used for immunohistochemistry.

Techniques: Migration, Transfection, Transwell Assay, Wound Healing Assay

Journal: Cancers

Article Title: Association of Proteasome Activity and Pool Heterogeneity with Markers Determining the Molecular Subtypes of Breast Cancer.

doi: 10.3390/cancers17010159

Figure Lengend Snippet: Figure 4. Proteasome activities and content in BC and adjacent tissues. Representative immunohis- tochemical staining of tumor tissues (a). First row (A1–A4) Invasive carcinoma of nonspecific type (Luminal A subtype): A1—Strong expression of ER; A2—Strong expression of PR; A3—negative

Article Snippet: Primary mouse monoclonal antibodies to 20S proteasome subunits α1, α2, α3, α5, α6, α7 (cat. # sc-58412), mouse mAbs to LMP-7 (cat. # sc-100284), rabbit polyclonal antibodies (pAbs) to LMP-2 (cat. # sc-28809), and goat antibodies to the subunit PA28β (cat. # sc-23642) were obtained from Santa Cruz Biotechnology, Dallas, TX, USA.

Techniques: Staining, Expressing

Journal: Cancers

Article Title: Association of Proteasome Activity and Pool Heterogeneity with Markers Determining the Molecular Subtypes of Breast Cancer.

doi: 10.3390/cancers17010159

Figure Lengend Snippet: Figure 5. Interrelation and mutual influence of proteasome activities and indicators on the basis of which the molecular subtype of breast cancer is determined. Note: →The positive influence of one characteristic on another.

Article Snippet: Primary mouse monoclonal antibodies to 20S proteasome subunits α1, α2, α3, α5, α6, α7 (cat. # sc-58412), mouse mAbs to LMP-7 (cat. # sc-100284), rabbit polyclonal antibodies (pAbs) to LMP-2 (cat. # sc-28809), and goat antibodies to the subunit PA28β (cat. # sc-23642) were obtained from Santa Cruz Biotechnology, Dallas, TX, USA.

Techniques: