ptyr  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: SRC-mediated phosphorylation of UBC9 regulates inflammatory and metabolic signaling in alcohol-associated liver disease

doi: 10.1126/sciadv.aec0138

Figure Lengend Snippet: ( A ) UBC9 mRNA ( n = 6 per group) and ( B ) protein levels from pair-fed and ethanol-fed mice livers ( n = 6 per group). Means ± SE. * P < 0.03 (mRNA) and * P < 0.04 (proteins) versus pair-fed (two-tailed t test). ( C ) Western blot analysis of phosphorylated UBC9 (pUBC9) and total UBC9 expression in isolated KCs and hepatocytes from NIAAA livers or pair-fed control livers. Means ± SE ( n = 3 per group). * P < 0.05 and ** P < 0.001 versus pair-fed (two-tailed t test). ( D ) Representative immunofluorescence images showing UBC9 phosphorylation in liver sections from pair-fed and ethanol-fed mice. Staining includes F4/80 (KCs marker; green), UBC9 (red), and pTyr PLA probe (far-red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 200 μm. Results are presented as means ± SE ( n = 3 per group). * P < 0.05 versus pair-fed (two-tailed t test). ( E ) Proximity ligation assay (PLA) demonstrating increased pUBC9 signal in CD68 + macrophages from human alcohol-associated steatohepatitis (ASH; n = 18) liver samples compared with normal livers ( n = 5), Scale bars, 200 μm. Data are presented as means ± SE ( n = 5 to 18). * P < 0.03 versus normal (two-tailed t test).

Article Snippet: Briefly, tissues were incubated with primary UBC9 (rabbit; Abcam, ab33044) and pTyr (mouse; Cell Signaling Technology, 9411S) or SRC (mouse; Abcam, ab231081) antibodies (1:150) overnight at 4°C in a humidity chamber after blocking.

Techniques: Two Tailed Test, Western Blot, Expressing, Isolation, Control, Immunofluorescence, Phospho-proteomics, Staining, Marker, Proximity Ligation Assay

Journal: Science Advances

Article Title: SRC-mediated phosphorylation of UBC9 regulates inflammatory and metabolic signaling in alcohol-associated liver disease

doi: 10.1126/sciadv.aec0138

Figure Lengend Snippet: Mouse hepatocytes were cultured with conditioned medium from LPS (500 ng/ml per 16 hours)– and/or PP1 (20 μM, 2 hours)–treated KCs and cotreated with ethanol (100 mM) for 24 hours. ( A ) Lipid accumulations were visualized by BODIPY staining ( n = 3 per group). Scale bars, 200 μm. ( B ) Triglycerides measurement in hepatocytes cultured with conditioned medium from LPS/PP1-treated KCs. Data are presented as means ± SE ( n = 3 per group). * P < 0.01 versus control, ** P < 0.05 versus control LPS, *** P < 0.01 versus control PP1, † P < 0.01 versus ethanol (EtOH) LPS, ‡ P < 0.0001 versus EtOH LPS (two-way ANOVA). ( C ) Schematic of in vivo PP1 treatment experimental design. H, hours. ( D ) Representative hematoxylin and eosin (H&E) and Oil Red O–stained liver sections from control, ethanol-fed, and PP1-treated mice. Scale bars, 200 μm ( n = 3 per group). ( E ) Plasma ALT and AST levels and liver triglyceride levels in ethanol-fed mice with or without PP1 treatment (1.5 mg/kg, every other day for 10 days). Means ± SE ( n = 3 per group). * P < 0.01 versus pair-fed, † P < 0.05 versus EtOH (AST; one-way ANOVA); * P < 0.0001 versus pair-fed, ** P < 0.01 versus pair-fed, † P < 0.01 versus EtOH (ALT; one-way ANOVA); * P < 0.001 versus pair-fed, † P < 0.02 versus EtOH (triglycerides; one-way ANOVA). ( F ) PLA images showing the physical interaction between UBC9 and SRC (red) in F4/80 + KCs (green) in liver sections from PP1-treated NIAAA model. DAPI was used as a counterstain (blue). Scale bars, 200 μm. PLA intensity per DAPI area was quantified by ImageJ. Means ± SE ( n = 3 per group). UBC9-SRC: * P < 0.01 versus pair-fed, † P < 0.01 versus EtOH (one-way ANOVA); UBC9-pTyr: ** P < 0.02 versus pair-fed, ‡ P < 0.05 versus EtOH (one-way ANOVA).

Article Snippet: Briefly, tissues were incubated with primary UBC9 (rabbit; Abcam, ab33044) and pTyr (mouse; Cell Signaling Technology, 9411S) or SRC (mouse; Abcam, ab231081) antibodies (1:150) overnight at 4°C in a humidity chamber after blocking.

Techniques: Cell Culture, Staining, Control, In Vivo, Clinical Proteomics