Gel pro analyzer software

Samples of aorta tissue were washed three times with cold PBS before being lysed in RIPA lysis buffer (10 mmol/L Tris-HCl, pH 7.5; 1% NP-40; 0.1% sodium deoxycholate; 0.1% SDS; 150 mmol/L NaCl; and 1 mmol/L EDTA) supplemented with 1× protease and phosphatase inhibitor cocktail (Thermo, Fremont, CA, USA) on ice. The separated proteins were transferred onto a nitrocellulose membrane. The anti-eNOS (1:100), anti-phospho-eNOS (1:100, Ser¹¹⁷⁷) and anti-β-actin (1:3000) antibodies and secondary antibodies (1:10000) were obtained from Cell Signaling Technology (Beverly, MA, USA). Immunoreactive protein bands were visualized using a ChemiDoc XRS+ System (Bio-Rad) and quantified with Gel Pro Analyzer software (Silk Scientific, Inc., Orem, UT, USA). The internal control, β-actin, was used to normalize differences due to loading variations.

Samples of liver tissue were washed three times with cold phosphate-buffered saline (PBS) before being lysed in radioimmunoprecipitation (RIPA) lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1% NP-40; 0.1% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl, and 1 mmol/L EDTA) supplemented with 1× protease and phosphatase inhibitor cocktail (Thermo, Fremont, CA, USA) on ice. The separated proteins were transferred onto a nitrocellulose membrane. The monoclonal anti-AMPK (1:100), anti-phospho-AMPK (1:200, Thr172), anti-HMGCR (1:100), anti-phospho-HMGCR (1:100, Ser872), anti-SREBP-2 (1:100), INSIG-1 (1:100), and anti-β-actin (1:3000) antibodies and secondary antibodies (1:10,000) were obtained from Abcam (Cambridge, MA, USA). Immunoreactivity protein bands were visualized using a ChemiDoc XRS+ System (Bio-Rad), and quantified with Gel Pro Analyzer software (Silk Scientific, Inc., Orem, UT, USA). The internal control, β-actin, was used to normalize differences due to loading variations.

Samples of epididymal or inguinal white adipose tissues were washed three times with cold PBS before being lysed in radioimmunoprecipitation (RIPA) lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1% NP-40; 0.1% sodium deoxycholate, 0.2% SDS, 150 mmol/L NaCl, and 1 mmol/L EDTA) supplemented with 1× protease and phosphatase inhibitor cocktail (Thermo, Fremont, CA, USA) on ice. The separated proteins were transferred onto a nitrocellulose membrane. The anti-ATGL (1:100, #2138), anti-HSL (1:200, #4107), anti-p-HSL (1:200. Ser563, #4139), anti-perilipin-1 (1:100, #3467), anti-UCP1 (1:100, #14670), anti-CPT1 (1:100), PGC1α (1:100, #4259), and anti-β-actin (1:3000, #4967) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The monoclonal anti-PPARα antibody (1:200, ab191226) and secondary antibodies (1:10,000) were obtained from Abcam (Cambridge, MA, USA). Immunoreactive protein bands were visualized using a ChemiDoc XRS+ System (Bio-Rad) and quantified with Gel Pro Analyzer software (Silk Scientific, Inc., Orem, UT, USA). The internal control, β-actin, was used to normalize differences due to loading variations.