Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: (A) Western blots of chondrocyte lysates, with densitometric analysis, revealed decreased PSMD11 in OA grade IV compared to normal donor cells. (B) PSMD11 mRNA levels were significantly decreased in OA compared to normal chondrocytes (p=0.0008, t-test). (C) Western blots, with densitometric analysis, also revealed decreased p-FOXO4 levels, but no consistent changes in total FOXO4, in OA compared to normal donor chondrocytes.

Article Snippet: Primary antibodies to PSMD11 (#14303), Caspase-3 (#9662), PARP (#9532) and HA-tag (#3724) were from Cell Signal Technologies (Danvers, MA).

Techniques: Western Blot

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: We studied PSMD11 siRNA knockdown effects on NO and MMP13 release in response to IL-1β (10 ng/ml) in normal human knee chondrocytes (A). We also analyzed PSMD11 siRNA effects on SOX9 and AGC1 (B), and SOX9 protein expression, as well as autophagy-related LC3A/B-I to LC3A/B-II conversion, the autophagy-UPS adaptor and LC3-binding protein p62 (C), and perinuclear co-localization of p62 and LC3A/B-I to LC3A/B-II in normal chondrocytes (D), with 300 cells/treatment counted to quantify structures where LC3A/B and p62 co-localized. Analysis was by 2-way ANOVA (Multiple comparison with Bonferroni post hoc comparison).

Article Snippet: Primary antibodies to PSMD11 (#14303), Caspase-3 (#9662), PARP (#9532) and HA-tag (#3724) were from Cell Signal Technologies (Danvers, MA).

Techniques: Knockdown, Expressing, Binding Assay, Comparison

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: (A) Transfecting Grade IV OA chondrocytes, we assessed effects of HA tag-PSMD11 plasmid, compared to control plasmid transfection, on proteasome activity (n=4 donors), and (B) on accumulation of K48-polyubiquitinated proteins (results representative of 4 donors). (C) PSMD11 gain of function by transfection in OA chondrocytes (n=5 different donors), validated to augment cellular HA, was assessed for effects on SOX9 levels. (D) Using ChIP assay, we separately analyzed the effect of PSMD11 gain of function on nuclear SOX9 binding to the AGC1 promoter and COL2A1 enhancer.

Article Snippet: Primary antibodies to PSMD11 (#14303), Caspase-3 (#9662), PARP (#9532) and HA-tag (#3724) were from Cell Signal Technologies (Danvers, MA).

Techniques: Plasmid Preparation, Control, Transfection, Activity Assay, Binding Assay

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: (A) Western blots of chondrocyte lysates, with densitometric analysis, revealed decreased PSMD11 in OA grade IV compared to normal donor cells. (B) PSMD11 mRNA levels were significantly decreased in OA compared to normal chondrocytes (p=0.0008, t-test). (C) Western blots, with densitometric analysis, also revealed decreased p-FOXO4 levels, but no consistent changes in total FOXO4, in OA compared to normal donor chondrocytes.

Article Snippet: Human PSMD11 cDNA was obtained from Addgene.org (pQTEV-PSMD11, ID 31333), amplified by PCR, and subcloned, with and without human influenza hemagglutinin (HA)-tagging at the N-terminal, into pCDNA3.1(+) plasmid vector between Xho1 and BamH1 sites (primers cited in Supplemental Table 1 ), and verification by sequencing.

Techniques: Western Blot

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: We studied PSMD11 siRNA knockdown effects on NO and MMP13 release in response to IL-1β (10 ng/ml) in normal human knee chondrocytes (A). We also analyzed PSMD11 siRNA effects on SOX9 and AGC1 (B), and SOX9 protein expression, as well as autophagy-related LC3A/B-I to LC3A/B-II conversion, the autophagy-UPS adaptor and LC3-binding protein p62 (C), and perinuclear co-localization of p62 and LC3A/B-I to LC3A/B-II in normal chondrocytes (D), with 300 cells/treatment counted to quantify structures where LC3A/B and p62 co-localized. Analysis was by 2-way ANOVA (Multiple comparison with Bonferroni post hoc comparison).

Article Snippet: Human PSMD11 cDNA was obtained from Addgene.org (pQTEV-PSMD11, ID 31333), amplified by PCR, and subcloned, with and without human influenza hemagglutinin (HA)-tagging at the N-terminal, into pCDNA3.1(+) plasmid vector between Xho1 and BamH1 sites (primers cited in Supplemental Table 1 ), and verification by sequencing.

Techniques: Knockdown, Expressing, Binding Assay, Comparison

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: (A) Transfecting Grade IV OA chondrocytes, we assessed effects of HA tag-PSMD11 plasmid, compared to control plasmid transfection, on proteasome activity (n=4 donors), and (B) on accumulation of K48-polyubiquitinated proteins (results representative of 4 donors). (C) PSMD11 gain of function by transfection in OA chondrocytes (n=5 different donors), validated to augment cellular HA, was assessed for effects on SOX9 levels. (D) Using ChIP assay, we separately analyzed the effect of PSMD11 gain of function on nuclear SOX9 binding to the AGC1 promoter and COL2A1 enhancer.

Article Snippet: Human PSMD11 cDNA was obtained from Addgene.org (pQTEV-PSMD11, ID 31333), amplified by PCR, and subcloned, with and without human influenza hemagglutinin (HA)-tagging at the N-terminal, into pCDNA3.1(+) plasmid vector between Xho1 and BamH1 sites (primers cited in Supplemental Table 1 ), and verification by sequencing.

Techniques: Plasmid Preparation, Control, Transfection, Activity Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/β-catenin pathway by inhibiting the proteasome

doi: 10.1074/jbc.M117.788448

Figure Lengend Snippet: PSMD2 is a substrate of UBE3A. A, cryo-EM structure of the proteasome showing the location of PSMD2 (green) in the 19S regulatory particle (Protein Data Bank code 5GJQ). B and C, PSMD2 dose-dependently rescues UBE3A-stimulated Wnt signaling. HEK293T cells were co-transfected with plasmids encoding UBE3AT485A and increasing quantities of PSMD2 (0, 20, 40, and 60 ng). Cells were then grown in L-cell CM (B) or Wnt3a CM (C). Mean percent values for firefly:Renilla ratios are shown relative to cells transfected with UBE3A + empty vector (n = 6). Error bars indicate S.D. ***, p < 0.0005, one-sample t test (two-tailed). D, HEK293T cells transfected with the indicated UBE3A, Myc-DDK-PSMD2, and HA-ubiquitin constructs were treated with the proteasome inhibitor MG-132 (30 μm; 4 h). PSMD2 was immunoprecipitated (IP) using an anti-FLAG antibody, and the Western blot was probed with an anti-HA antibody (left panel) or PSMD2 antibody (right panels) to detect ubiquitinated PSMD2. E, an in vitro ubiquitin assay was performed using recombinant E1, E2 (UBE2D3), UBE3A, and PSMD2 expressed and purified from HEK293T cells. Reactions were stopped at the indicated time, and the formation of ubiquitinated PSMD2 was monitored using a PSMD2 antibody. WT UBE3A was omitted from the reaction as a negative control (first lane).

Article Snippet: Ub G76V -GFP was obtained from Addgene (23969). pCMV6 Myc-DDK-tagged constructs for PSMB1 (RC201798), PSMC2 (RC200945), PSMC5 (RC201251), PSMC6 (RC202809), PSMD1 (RC210486), PSMD2 (RC203204), PSMD3 (RC202307), PSMD6 (RC202292), PSMD7 (RC203133), PSMD11 (RC201201), and untagged PSMD4 (SC111678) were all purchased from Origene.

Techniques: Cryo-EM Sample Prep, Transfection, Plasmid Preparation, Two Tailed Test, Construct, Immunoprecipitation, Western Blot, In Vitro, Ubiquitin Assay, Recombinant, Purification, Negative Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: We studied PSMD11 siRNA knockdown effects on NO and MMP13 release in response to IL-1β (10 ng/ml) in normal human knee chondrocytes (A). We also analyzed PSMD11 siRNA effects on SOX9 and AGC1 (B), and SOX9 protein expression, as well as autophagy-related LC3A/B-I to LC3A/B-II conversion, the autophagy-UPS adaptor and LC3-binding protein p62 (C), and perinuclear co-localization of p62 and LC3A/B-I to LC3A/B-II in normal chondrocytes (D), with 300 cells/treatment counted to quantify structures where LC3A/B and p62 co-localized. Analysis was by 2-way ANOVA (Multiple comparison with Bonferroni post hoc comparison).

Article Snippet: Primary antibodies to PSMG1 (#13378), PSMD11 (#14303), FOXO1 (#2880), FOXO3a (#12829), FOXO4 (#2499), phosphorylated FOXO1/FOXO3a (p-FOXO1/p-FOXO3A) (#9464), Sox9 (#82630), LC3/A/B (#12741), p62 (#8025), and HA-tag (#3724) were from Cell Signal Technologies (Danvers, MA).

Techniques: Knockdown, Expressing, Binding Assay, Comparison

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels

doi: 10.1002/art.40456

Figure Lengend Snippet: (A) Transfecting Grade IV OA chondrocytes, we assessed effects of HA tag-PSMD11 plasmid, compared to control plasmid transfection, on proteasome activity (n=4 donors), and (B) on accumulation of K48-polyubiquitinated proteins (results representative of 4 donors). (C) PSMD11 gain of function by transfection in OA chondrocytes (n=5 different donors), validated to augment cellular HA, was assessed for effects on SOX9 levels. (D) Using ChIP assay, we separately analyzed the effect of PSMD11 gain of function on nuclear SOX9 binding to the AGC1 promoter and COL2A1 enhancer.

Article Snippet: Primary antibodies to PSMG1 (#13378), PSMD11 (#14303), FOXO1 (#2880), FOXO3a (#12829), FOXO4 (#2499), phosphorylated FOXO1/FOXO3a (p-FOXO1/p-FOXO3A) (#9464), Sox9 (#82630), LC3/A/B (#12741), p62 (#8025), and HA-tag (#3724) were from Cell Signal Technologies (Danvers, MA).

Techniques: Plasmid Preparation, Control, Transfection, Activity Assay, Binding Assay

Journal: Communications Biology

Article Title: The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells

doi: 10.1038/s42003-020-0984-3

Figure Lengend Snippet: a In vitro ubiquitination assay of 6xHis-tagged H3F3A with UBE2K and FLAG::RNF2 or GFP::RNF138 ubiquitin ligases followed by immunoblotting with antibodies to 6xHis, UBE2K, FLAG and GFP. The images are representative of two independent experiments. b In vitro ubiquitination of recombinant p53 followed by immunoblotting with antibodies to p53, UBE2K, and GFP. The images are representative of two independent experiments. c In vitro ubiquitination of 6xHis::H1 followed by immunoblotting with antibodies to 6xHis, UBE2K, and GFP. The images are representative of two independent experiments. d Western blot of UBE2K overexpressing (OE) HEK293 with antibodies to H3, H3K9me3 and β-actin. The graphs represent the relative percentage of H3/β-actin and H3K9me3/H3 to DMSO-empty vector cells (mean ± s.e.m. of four independent experiments). When indicated in the figure, cells were treated with 5 µM MG-132 for 16 h. e Knockdown levels of proteasome activators in HEK293 cells. The graph (relative expression to non-targeting (NT) shRNA HEK293 cells) represents the mean ± s.e.m. (PSMD11 shRNA (n = 8), PSME4 shRNA ( n = 5), PSME3 shRNA ( n = 6)). f Percentage of chymotrypsin-like proteasome activity relative to NT shRNA HEK293 cells (mean ± s.e.m. of three independent experiments). MG-132 treatment: 5 µM MG-132 for 16 h. g Western blot of HEK293 cells with antibodies to H3 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA cells (mean ± s.e.m. of three independent experiments). h Western blot of HEK293 cells with antibodies to H3, PSMD11 and UBE2K. The graph represents the relative percentage of H3/β-actin to NT shRNA + empty vector cells (mean ± s.e.m. of three independent experiments). i After immunoprecipitation with anti-H3 and anti-FLAG antibodies in HEK293 cells, we performed a re-immunoprecipitation (Re-IP) with the same antibodies. Re-IP was followed by western blot with antibodies against H3 and polyubiquitinated proteins (polyUb) to detect immunoprecipitated H3 protein and polyUb-H3, respectively. The images are representative of two independent experiments. Prior to immunoprecipitation, cells were treated with 5 µM MG-132 (16 h). All the statistical comparisons were made by two-tailed Student’s t -test for unpaired samples. P value: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS not significant.

Article Snippet: Western blot analysis was performed with anti-UBE2K (Cell Signaling, #8226, 1:1,000), anti-OCT4 (Stem Cell Technologies, #60093, 1:500), anti-SOX2 (Abcam, #97959, 1:1,000), anti-PAX6 (Stem Cell Technologies, #60094, 1:200), anti-Nestin (Stem Cell Technologies, #60091, 1:1,000), anti-MAP2 (Sigma, #1406, 1:1,000), anti-polyubiquitinylated conjugates (Enzo, PW8805-0500, 1:1,000), anti-ubiquitin (Merck Millipore, # 05-944, clone P4D1-A11, 1:1000), anti-H3K9me3 (Abcam, #8898, 1:1,000), anti-Histone H3 (Cell Signaling, #2650, 1:10,000), anti-H3K9me1 (Cell Signaling, #1418, 1:1,000), anti-H3K9me2 (Cell Signaling, #4658, 1:1,000), anti-H3K4me3 (Active Motif, #39916, 1:1,000), anti-H3K27me3 (Active Motif, #39155, 1:1,000), anti-H3K27ac (Active Motif, #39933, 1:1,000), anti-HTT (Cell Signaling, #5656, 1:1,000), anti-SETDB1 (Abcam, #107225, 1:500), anti-p53 (Cell Signaling, #9282, 1:2,000), anti-Histone H1 (Merck, 05-457, 1:1,000), anti-PSMD11 (Abcam, #99413, 1:1,000), anti-ß-actin (Abcam, #8226, 1:1,000) and α-tubulin (Sigma, T6199, 1:5,000).

Techniques: In Vitro, Ubiquitin Assay, Western Blot, Recombinant, Plasmid Preparation, Expressing, shRNA, Activity Assay, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: TRIM11 activates the proteasome and promotes overall protein degradation by regulating USP14

doi: 10.1038/s41467-018-03499-z

Figure Lengend Snippet: TRIM11 activates the proteasome. a Overall proteolysis in control and TRIM11-overexpressing HCT116 cells treated with BTZ (0.5 μM) or CQ (50 μM). b Levels of K48 polyUb conjugates in wild-type and Atg5 KO MEF infected with vector or TRIM11 lentiviruses, and grown under normal or heat stress conditions. c , d Chymotrypsin-like proteasome activity in TRIM11-overexpressing HCT116 ( c ), TRIM11-depleted HCT116 cells ( d ), and the corresponding control cells, as measured by fluorometric substrate Suc-LLVY-AMC. In c , cells were also treated with BTZ (200 nM, 4 h). Slopes relative to that of control are shown, n = 7 ( c , without BTZ), 6 ( c , with BTZ), and 8 ( d ). e Chymotrypsin-like proteasome activity in control and TRIM11-overexpressing HCT116 cells cultured under unstressed, heat shock, and heat shock/recovery conditions. Slopes relative to that of control are shown, n = 7. In a and c – e , data represent the mean ± SEM ( n = 3, unless otherwise indicated). * P < 0.05; ** P < 0.01; *** P < 0.001. Uncropped blots are presented in Supplementary Fig. . n.s.: not significant

Article Snippet: Primary antibodies against the following epitope or proteins were purchased from the indicated sources: tubulin (T6074, 1:2000), Flag M2 (F3165, 1:50), and Flag M2 magnetic beads (Sigma, 1:50); HA (71-5500, 1:1000) and HA agarose (Thermo Fisher Scientific, 1:50); GFP (M048-3, 1:1000) (MBL); GAPDH (NB300-221, 1:3000) (Novus Biologicals); TRIM11 (ABC926, 1:500) (EMD Millipore); 20S proteasome α/β subunits (BML-PW8155, 1:1000) and 20S proteasome α1–7 (BML-PW8195, 1:1000) (Enzo Life Sciences); p53 (DO-1, sc-126, 1:2000), p21 (sc-6246, 1:500), Mdm2 (sc-965, 1:500), ubiquitin (P4D1, sc-8017, 1:2000), USP14 (6E6, sc-100630, 1:1000) and HSF1 (H-311, sc-9144, 1:2000) (Santa Cruz Biotechnology); caspase-3 (9662, 1:1000), cleaved caspase-3 (9661, 1:1000), ATG5 (2630, 1:1000), and K48-linked polyubiquitin chain (4289, 1:1000) (Cell Signaling Technology); and USP14 (A300-919A, 1:1000), TRIM28 (A300-275A, 1:2000), PSMD1 (A303-851A, 1:1000), PSMD2 (A303-853A, 1:1000), and PSMD11 (A302-750A, 1:500) (Bethyl Laboratories).

Techniques: Control, Infection, Plasmid Preparation, Activity Assay, Cell Culture