expression plasmid construction human psmd11 cdna  (Addgene inc)

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: Transfection and expression plasmid construction 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: Transfection and expression plasmid construction 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: Transfection and expression plasmid construction 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