Journal: Lipids in health and disease

Article Title: Polysaccharide CM1 from Cordyceps militaris hinders adipocyte differentiation and alleviates hyperlipidemia in LDLR (+/-) hamsters.

doi: 10.1186/s12944-021-01606-6

Figure Lengend Snippet: Fig. 3 Effect of CM1 on the expression of lipid metabolism-related genes in the liver of LDLR(+/−) hamsters (n = 3). A, B, C, and D, mRNA expression of SREBP-2, PCSK9, LXRα, and SREBP-1c, respectively

Article Snippet: Rabbit polyclonal antibodies were used against PCSK9 (55206–1-AP), albumin (16475–1- AP), and apoB (20578–1-AP) (Proteintech, IL, USA).

Techniques: Expressing

Journal: Lipids in health and disease

Article Title: Polysaccharide CM1 from Cordyceps militaris hinders adipocyte differentiation and alleviates hyperlipidemia in LDLR (+/-) hamsters.

doi: 10.1186/s12944-021-01606-6

Figure Lengend Snippet: Fig. 4 Effect of CM1 on the expression of TC metabolism-related proteins in the liver of the LDLR(+/−) hamsters (n = 5). A, SR-B1; B, LDLR; C, SREBP-2; D, PCSK9; E, CYP7A1; F, ABCG8; G, ABCG5; H, LXRα expression and densitometric quantification

Article Snippet: Rabbit polyclonal antibodies were used against PCSK9 (55206–1-AP), albumin (16475–1- AP), and apoB (20578–1-AP) (Proteintech, IL, USA).

Techniques: Expressing

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) SAR1B nitrosylates SURF4. FLAG-tagged SNO-SAR1B was incubated with V5-tagged SURF4. Reaction mixtures were subjected to SNO-RAC and SNO-proteins visualized by western blot. Representative image (n = 3) is shown. (B) SURF4 nitrosylates PCSK9. V5-tagged SNO-SURF4 was incubated with FLAG-tagged PCSK9. Reaction mixtures were subjected to SNO-RAC and SNO-proteins visualized by western blot. Representative image (n = 3) is shown. Mature PCSK9 is visualized in SNO-PCSK9 lanes. (C) Western blot analysis of SNO-PCSK9 and SNO-SAR1B in SCoR2-deficient HEK293 transfected with wild-type PCSK9 and wild-type SAR1B and treated with 200 μM ethyl ester S-nitroso-cysteine (ECySNO) for 90 min. Anti-FLAG antibody was used to visualize SAR1B. (D) Quantification (n = 3) of SNO-PCSK9 and SNO-SAR1B (normalized to total PCSK9 and SAR1B, respectively) from (C) and related experiments. (E) Western blot analysis of SNO-SAR1B wild-type and indicated mutations in SCoR2-deficient HEK293 transfected with SAR1B wild-type and indicated mutations and treated with 200 μM ECySNO for 90 min. Anti-FLAG antibody was used to visualize SAR1B in a single experiment that is verified in subsequent assays. (F) Western blot analysis of SNO-PCSK9, SNO-SURF4, and SNO-SAR1B in SCoR2-deficient HEK293 cells transiently overexpressing SAR1B WT or SAR1B C102A/C178A and treated with 200 μM ECySNO for 90 min prior to harvest. (G) Quantification (n = 3) of SNO-PCSK9 (mature band, normalized to total mature PCSK9) and SNO-SURF4 from (F). (H) Representative western blot analysis for cellular and secreted (media) PCSK9 in SCoR2-deficient HEK293 cells overexpressing SAR1B WT or SAR1B C102A/C178A and treated with 200 μM ECySNO for 90 min prior to harvest. (I) Quantification (n = 3) of secreted (media) PCSK9 (normalized to mature PCSK9 band) from (H). p values in (I) were calculated by one-way ANOVA. (J) SCoR-deficient HEK293 cells stably expressing PCSK9 were treated with or without 200 μM ECySNO (+SNO) for 90 min then stained with anti-PCSK9 (green) and anti-calnexin (red, ER marker) antibodies. Scale bar, 5 μm. (K) Quantification of mean PCSK9 signal intensity in pixels positive for calnexin (n = 12 cells per condition). Control: SNO-RAC assay performed without ascorbate. See also .

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Incubation, Western Blot, Transfection, Stable Transfection, Expressing, Staining, Marker, Control

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet:

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Recombinant, Cholesterol Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, shRNA, Mutagenesis, Software