Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 1. Comparison of NPRA and NPRC protein expression. (A) Protein expression of NPRA and NPRC were visualized by western blotting in membrane enriched fractions prepared from a variety of mouse tissues: gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), heart, liver, kidney, and lung. (B) BAT membrane enriched fractions from wildtype and NPRC−/− mice fed either standard chow or HFD were used to visualize NPRA and NPRC protein levels. Šídák’s multiple comparisons test for two-way ANOVA indicated on graph. * P < 0.05, *** P < 0.001, **** P < 0.0001. (C) BAT protein from chow- and HFD-fed mice was separated into membrane and cytosolic enriched fractions. NPRA and NPRC were assessed by western blotting. (D) HIB-1B cells were transfected with the indicated plasmids and treated with adipocyte differentiation cocktail for 48 h. Cells were then treated with the 2.5 µM MG-132 for 16 h. Flag-NPRA and NPRC were assessed from whole-cell lysates by western blotting. Šídák’s multiple comparisons test for one-way ANOVA indicated on graph. ** P < 0.01, *** P < 0.001.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Comparison, Expressing, Western Blot, Membrane, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 2. NPRC immunoprecipitates with NPRA and NPRB. HEK 293FT cells were transfected with plasmids expressing Flag-tagged natriuretic peptide receptors. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag Affinity agarose. (A) Endogenous NPRC coimmunoprecipitated with hNPRA-Flag, hNPRB-Flag, or VEGFR1-Flag. (B) Endogeneous NPRC coimmunoprecipitated with GC-C-Flag. (C) NPRC band intensity to input of NPRC was calculated through Image J software was presented in the right panel. Šídák’s multiple comparisons test for one-way ANOVA comparing all samples to the negative control VEGFR1 for western blots in A and B are indicated on graph. *P < 0.05, ** P < 0.01, *** P < 0.001. (D) hNPRA-Flag expressing HEK 293FT cells were treated with ANP (ANP1-28, 200 nM) or a truncated form of ANP that preferentially binds NPRC (ANP4-23, 200 nM) for 30 min. Neither ANP1-28 or ANP4-23 affected the ability of endogenous NPRC to coimmunoprecipitate with hNPRA-Flag. (E) Endogenous NPRC coimmunoprecipitated with Flag-rNPRA and a truncated version of Flag- rNPRA lacking the c-terminal amino acids 528-1057 [Flag-rNPRA(1-527)].

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Transfection, Expressing, Immunoprecipitation, Software, Negative Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 3. Endogenous natriuretic peptide receptor expression in cell models used. Expression of NPRA, NPRB, and NPRC were detected by western blot in HeLa, HEK293FT, and HAP-1 cells. β-actin was used as the loading control.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Expressing, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 6. Coexpression of NPRC reduces ANP-evoked cGMP production in plasma membranes. Plasma membranes were made from HEK-293T cells transfected with either NPRA-flag plus pcDNA3-Clover control or NPRA-flag plus NPRC-flag. Plasma membranes were made without or with (SI Appendix, Fig. S3) phosphatase inhibitors. Co-expression of hNPRC along with hNPRA reduced the cGMP generated in the presence of 100 nM ANP (P = 0.0163, test for difference in slopes between NPRA and NPRA+NPRC; Šídák’s multiple comparisons test comparing NPRA vs. NPRA+NPRC are indicated on the graphs, ** P < 0.01.). Untransfected cells did not appear to generate cGMP. Western blot of membranes used in the ANP-stimulated cGMP assay.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Clinical Proteomics, Transfection, Control, Expressing, Generated, 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: (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: 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