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