anti glut1  (Alomone Labs)

Journal: Glia

Article Title: Glial hypothalamic inhibition of GLUT2 expression alters satiety, impacting eating behavior, et al. Glial hypothalamic inhibition of GLUT2 expression alters satiety, impacting eating behavior

doi: 10.1002/glia.23267

Figure Lengend Snippet: Injection of Ad‐shGLUT2 into the 3V inhibits GLUT2 without altering GK and GLUT1. (a) Experimental protocol. (b‐d) Analysis of GLUT2, GK, and GLUT1 mRNA expression relative to cyclophilin in hypothalamic samples from rats transduced with Ad‐shβgal or Ad‐shGLUT2 by qRT‐PCR. (e) Western blot analysis of hypothalamic protein extracts isolated from rats transduced with Ad‐shβgal (lane 1) or Ad‐shGLUT2 (lane 2) for 48 hr. (f‐h) Densitometric analysis of each protein relative to β‐actin evaluated in Ad‐shGLUT2‐injected rats, as a percentage of levels observed in Ad‐shβgal‐EGFP‐injected rats. Results are representative of four independent experiments performed in triplicate. Data are expressed as mean ±SD. * p

Article Snippet: Samples were homogenized in buffer A (0.3 mM sucrose, 3 mM DTT, 1 mM EDTA, 100 µg/ml PMSF, 2 µg/ml pepstatin A, 2 µg/ml leucopeptin, and 2 mg/ml aprotinin), sonicated three times on ice at 60 Hz for 10 s (Sonics & Material INC, VCF1, Newtown, CT, USA), and separated by centrifugation at 8,000g for 10 min. Proteins were resolved by SDS‐PAGE (50 µg/lane) in a 12% (w/v) polyacrylamide gel, transferred to PVDF membranes (0.45 mm pore, Amersham Pharmacia Biotech., Piscataway, NJ, USA), and probed for 16 hr at 4°C with rabbit anti‐GLUT2 (1:1,000; Alpha Diagnostic, San Antonio, TX, USA), anti‐GLUT1 (1:1,000; Alomone Labs, Jerusalem, Israel), anti‐glucokinase (GK; 1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti‐β‐actin (1:10,000; Santa Cruz).

Techniques: Injection, Expressing, Transduction, Quantitative RT-PCR, Western Blot, Isolation