anti mc5 r  (Alomone Labs)

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: ( A ) Immunostaining of MC1-R staining in the liver of chow-fed C57Bl/6 J mouse. In the control section, anti-MC1-R antibody was replaced by purified normal rabbit IgG (isotype control). Scale bar, 50 µm. ( B ) Immunofluorescence staining of MC1-R (red) and the hepatocyte marker serum albumin (green) in the liver of chow-fed C57Bl/6 J mouse. Scale bar, 20 μm. ( C ) Quantitative real-time polymerase chain reaction (qPCR) analysis of Mc1r mRNA expression in the liver of chow- and Western diet-fed mice. ( D ) Representative Western blots of MC1-R and β-actin (loading control) and quantification of MC1-R protein level in the liver of chow- and Western diet-fed mice. *p <0.05 and **p <0.01 versus chow-fed mice by Student’s t-test. ( E ) MC1R gene expression in human liver biopsies from control cases (n=10) and patients with nonalcoholic fatty liver disease (NAFLD, n=51) or nonalcoholic steatohepatitis (NASH, n=155). Violin plots show normalized log2 RPKM values (reads per kilobase of exon per million reads mapped) and medians (dashed line) for each sample group. ( F ) Schematic presentation of the loxP-flanked (floxed) Mc1r allele and the positions of forward and reverse primers used for PCR genotyping. PCR analysis of genomic DNA extracted from the liver of Alb-Cre-negative and -positive mice that were homozygous for the Mc1r floxed allele ( Mc1r fl/fl ). The size of the recombined allele is ~217 bp. ( G ) qPCR analysis of Mc1r expression in the liver of chow-fed Mc1r fl/fl , Mc1r fl/+ Alb Cre/+ , and Mc1r LKO ( Mc1r fl/fl Alb Cre/+ ) mice at the age of 16 weeks. ( H, I ) Absolute liver weight and liver to body weight ratio (expressed as a percentage of body weight) in chow-fed Mc1r fl/fl , Mc1r fl/+ Alb Cre/+ , and Mc1r LKO mice at the age of 16 weeks. Values are mean ± SEM, n=5–10 mice per group in each graph. *p <0.05 , **p <0.01, and ****p <0.0001 for the indicated comparisons by one-way ANOVA and Dunnet post hoc tests. Mc1r LKO, hepatocyte-specific MC1-R knock-out mice. Figure 1—source data 1. Uncropped Western blots for .

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Immunostaining, Staining, Control, Purification, Immunofluorescence, Marker, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Knock-Out

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: ( A ) Immunofluorescence staining of MC1-R (red) and the cholangiocyte marker cytokeratin 19 (CK19, green) in the liver of a chow-fed C57Bl/6 J mouse. ( B ) Immunofluorescence staining of MC1-R (red) and the endothelial cell marker CD31 (green) in the liver of a chow-fed C57Bl/6 J mouse. ( C ) Immunofluorescence staining of MC1-R (red) and the macrophage marker Mac-2 (green) in the liver of a chow-fed C57Bl/6 J mouse. White arrows indicate co-localization of MC1-R and CK-19 or Mac-2. Scale bar, 20 μm in all images.

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Immunofluorescence, Staining, Marker

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: Western blot analysis of MC1-R protein expression in the mouse liver, HepG2, and mouse heart samples. The expression of β-actin is shown as a loading control. Lanes on the right (same samples as on the left) were incubated in an anti-MC1-R antibody solution that was premixed with a molar excess of a blocking MC1-R peptide. Figure 1—figure supplement 2—source data 1. Uncropped Western blots for .

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Western Blot, Expressing, Control, Incubation, Blocking Assay

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: ( A ) Representative Western blots of MC1-R and vinculin (loading control) and ( B ) quantification of MC1-R protein level in the liver of chow-fed control ( Mc1r fl/+ Alb Cre/+ ) and Mc1r LKO mice. *p<0.05 versus control mice by Student’s t-test. ( C ) Body weight curves of chow-fed Mc1r fl/f l , Mc1r fl/+ Alb Cre/+ , and Mc1r LKO mice. ( D and E ) Total fat and lean mass of chow-fed Mc1r fl/f l , Mc1r fl/+ Alb Cre/+ , and Mc1r LKO mice at the start and end of the body weight monitoring period. The change in fat and lean mass between the start and end of the experiment is also presented in the graphs. Values are mean ± SEM, mice n=5–10 mice per group in each graph. Figure 1—figure supplement 3—source data 1. Uncropped Western blots for .

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Western Blot, Control

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: ( A–C ) Representative Western blots and quantification of MC1-R protein level in HepG2 cells treated with palmitic acid (500 µM), LDL (200 µg/ml), or atorvastatin (10 µM) for 1, 3, 6, or 24 hr. ( D ) Quantification of free cholesterol content using filipin staining in HepG2 cells treated with α-MSH (1 µM) for 1, 3, 6, or 24 hr. ( E ) Quantification of free cholesterol content in HepG2 cells treated with different concentrations of α-MSH (0.1 nM, 10 nM, or 1 µM) for 24 hr. ( F, G ) Quantification of LDL and HDL uptake in HepG2 cells treated with different concentrations (0.1 nM, 10 nM, or 1 µM) of α-MSH for 24 hr. ( H, I ) Quantitative real-time polymerase chain reaction (qPCR) analysis of LDL receptor ( LDLR) and SCARB1 expression in HepG2 cells treated with different concentrations of α-MSH for 3, 6, or 24 hr. ( J, K ) Representative Western blots and quantification of LDL-R and SR-BI proteins levels in HepG2 cells treated with 1 µM α-MSH for 1, 3, 6, or 24 hr. ( L ) Quantification of cell surface LDLR by flow cytometry in HepG2 cells treated with 1 µM α-MSH for 24 hr. Values are mean ± SEM, n=3–6 per group in each graph. *p<0.05 and **p <0.01 for the indicated comparisons by one-way ANOVA and Dunnet post hoc tests ( A–K ) or by Student’s t-test ( L ). Figure 5—source data 1. Uncropped Western blots for .

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Western Blot, Staining, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: ( A ) Quantification of free cholesterol content using filipin staining in HepG2 cells treated with different concentrations of the selective MC1-R agonist LD211 (0.1 nM, 10 nM, or 1 µM) for 24 hr. ( B, C ) Quantification of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) uptake in HepG2 cells treated with different concentrations (0.1 nM, 10 nM, or 1 µM) of LD211 for 24 hr. ( D ) Quantitative real-time polymerase chain reaction (qPCR) analysis of LDL receptor ( LDLR) and SCARB1 expression in HepG2 cells treated with 1 µM LD211 for 3, 6, or 24 hr. ( E ) Representative Western blots and quantification of LDL-R and SR-BI proteins levels in HepG2 cells treated with 1 µM LD211 for 1, 3, 6, or 24 hr. ( F ) Quantification of cell surface LDLR by flow cytometry in HepG2 cells treated with 1 µM LD211 for 24 hr. Values are mean ± SEM, n=3–6 per group in each graph. *p<0.05 , **p<0.01 , ***p<0.001 , and ****p<0.0001 for the indicated comparisons by one-way ANOVA and Dunnet post hoc tests. Figure 6—source data 1. Uncropped Western blots for .

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry

Journal: eLife

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.7554/eLife.84782

Figure Lengend Snippet: Hepatocyte-specific MC1-R deficiency enhanced the accumulation of cholesterol and triglycerides (TG) in the liver, promoted fibrogenesis, and lead to a disturbance in bile acid metabolism. Conversely, activation of MC1-R with the endogenous agonist α-MSH or the synthetic agonist LD211 reduced cellular cholesterol levels and increased the uptake of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles in cultured HepG2 cells. p-AMPK indicates phosphorylated AMP-activated protein kinase; p-ERK1/2, phosphorylated extracellular-signal-regulated kinase 1/2; p-JNK, phosphorylated c-Jun N-terminal kinase. The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

Article Snippet: After blocking with Tris-Buffered Saline (Sigma-Aldrich) containing 0.1% Tween 20 detergent (Sigma-Aldrich) and 5% skimmed milk (Carl Roth) for 1 hr at room temperature (RT), membranes were incubated with specific primary antibodies for MC1-R (Alomone Labs, #AMR-025), LDLR (Novus Biologicals, Littleton, CO, USA, #NBP1-06709), SR-BI (NovusBio, #NB400-104), SREBP2 (Novus Biologicals, #NB100-74543), HMGCR (Novus Biologicals, #NBP2-66888), DHCR7 (abcam, #ab103296), MRP4 (Cell Signaling Tech, Frankfurt, DE, #12857), StAR (Cell Signaling Tech, #8449), CYP8B1 (St John’s Laboratory Ltd, #STJ92607), phospho-AMPKα (Cell Signaling Tech, #2535), and AMPKα (Cell Signaling Tech, #2532) over-night at +4 °C.

Techniques: Activation Assay, Cell Culture, Generated

Journal: bioRxiv

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.1101/2022.11.08.515543

Figure Lengend Snippet: ( A) Immunostaining of MC1-R staining in the liver of chow-fed C57Bl/6J mouse. In control section, anti MC1-R antibody was replaced by purified normal rabbit IgG (isotype control). Scale bar, 50 μm. ( B) Quantitative real-time polymerase chain reaction (qPCR) analysis of Mc1r mRNA expression in the liver of chow- and Western diet-fed mice. ( C) Representative Western blots of MC1-R and β-actin (loading control) and quantification of MC1-R protein level in the liver of chow- and Western diet-fed mice. ( D) Schematic presentation of the loxP-flanked (floxed) Mc1r allele and the positions of forward and reverse primers used for PCR genotyping. PCR analysis of genomic DNA extracted from the liver of Alb-Cre-negative and -positive mice that were homozygous for the Mc1r floxed allele (Mc1r fl/fl ). The size of the recombined allele is ∼217 bp. ( E ) qPCR analysis of Mc1r expression in the liver of chow-fed Mc1r fl/fl , L-Mc1r +/- and L-Mc1r -/- mice at the age of 16 weeks. ( F, G ) Absolute liver weight and liver to body weight ratio (expressed as percentage of body weight) in chow-fed Mc1r fl/fl , L-Mc1r +/- and L-Mc1r -/- mice at the age of 16 weeks. Values are mean ± SEM, n = 5-10 mice per group in each graph. * P<0 . 05 and ** P<0 . 01 for the indicated comparisons by one-way ANOVA and Dunnet post hoc tests. L-Mc1r +/- indicates Alb-Cre +/- mice that were heterozygous for the Mc1r floxed allele; L-Mc1r -/- , hepatocyte-specific MC1-R knock-out mice.

Article Snippet: After blocking, sections were incubated overnight with a primary antibody against MC1-R (Alomone Labs, Jerusalem, Israel, # AMR-025) followed by biotinylated horseradish peroxidase-conjugated secondary antibody incubation and detection with diaminobenzidine (ABC kit, Vector Labs, Burlingame, USA).

Techniques: Immunostaining, Staining, Purification, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.1101/2022.11.08.515543

Figure Lengend Snippet: ( A-C ) Representative Western blots and quantification of MC1-R protein level in HepG2 cells treated with palmitic acid (500 μM), LDL (200 μg/ml) or atorvastatin (10 μM) for 1, 3, 6 or 24 hours. (D) Quantification of free cholesterol content using filipin staining in HepG2 cells treated with α-MSH (1 μM) for 1, 3, 6 or 24 hours. (E) Quantification of free cholesterol content in HepG2 cells treated with different concentrations of α-MSH (0.1 nM, 10 nM or 1 μM) for 24 hours. (F, G) Quantification of LDL and HDL uptake in HepG2 cells treated with different concentrations (0.1 nM, 10 nM or 1 μM) of α-MSH for 24 hours. (H, I) qPCR analysis of LDLR and SCARB1 expression in HepG2 cells treated with different concentrations of α-MSH for 3, 6 or 24 hours. (J, K) Representative Western blots and quantification of LDL-R and SR-BI proteins levels in HepG2 cells treated with 1μM α-MSH for 1, 3, 6 or 24 hours. ( L ) Quantification of cell surface LDLR by flow cytometry in HepG2 cells treated with 1μM α-MSH for 24 hours. Values are mean ± SEM, * P<0 . 05 and ** P<0 . 01 for the indicated comparisons by one-way ANOVA and Dunnet post hoc tests ( A-K ) or by Student’s t test ( L ).

Article Snippet: After blocking, sections were incubated overnight with a primary antibody against MC1-R (Alomone Labs, Jerusalem, Israel, # AMR-025) followed by biotinylated horseradish peroxidase-conjugated secondary antibody incubation and detection with diaminobenzidine (ABC kit, Vector Labs, Burlingame, USA).

Techniques: Western Blot, Staining, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver

doi: 10.1101/2022.11.08.515543

Figure Lengend Snippet: (A) Quantification of free cholesterol content using filipin staining in HepG2 cells treated with different concentrations of the selective MC1-R agonist LD211 (0.1 nM, 10 nM or 1 μM) for 24 hours. (B, C) Quantification of LDL and HDL uptake in HepG2 cells treated with different concentrations (0.1 nM, 10 nM or 1 μM) of LD211 for 24 hours. (D) qPCR analysis of LDLR and SCARB1 expression in HepG2 cells treated with 1 μM LD211 for 3, 6 or 24 hours. (E) Representative Western blots and quantification of LDL-R and SR-BI proteins levels in HepG2 cells treated with 1μM LD211 for 1, 3, 6 or 24 hours. ( F ) Quantification of cell surface LDLR by flow cytometry in HepG2 cells treated with 1μM LD211 for 24 hours. Values are mean ± SEM, * P<0 . 05 , ** P<0 . 01 , *** P<0 . 001 and **** P<0 . 0001 for the indicated comparisons by one-way ANOVA and Dunnet post hoc tests.

Article Snippet: After blocking, sections were incubated overnight with a primary antibody against MC1-R (Alomone Labs, Jerusalem, Israel, # AMR-025) followed by biotinylated horseradish peroxidase-conjugated secondary antibody incubation and detection with diaminobenzidine (ABC kit, Vector Labs, Burlingame, USA).

Techniques: Staining, Expressing, Western Blot, Flow Cytometry