Xbt 1 film

Total proteins were extracted from kidney and renal tubules with RIPA buffer. 100 μg protein were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked at room temperature for 1 h in TBST (Tris buffered saline containing 0.1% Tween 20) containing 5% skimmed milk and was then incubated with primary antibodies at 4°C overnight. The membrane was washed for 5 min with TBST buffer for 3–5 times and then incubated with a horseradish peroxide-conjugated secondary antibody at a dilution 1:4000 at room temperature for 1 h. After washing, the membrane was developed with ECL Reagent and exposed to Kodak XBT-1 film. Anti-TMOD1 antibody was prepared by AbMax Biotechnology Co., Ltd. (Beijing, China). Anti-GAPDH antibody was purchased from Santa Cruz Biotech. (Santa Cruz, CA). Antibodies for TGFBR2, SLC25A11, MTFP1 were from Abclonal (Wuhan, China).

Western blot was performed as described previously [10 (link)]. Briefly, proteins from tissues or cells were extracted and quantified. Aliquots of 10 μg total protein of each sample were separated by 8%-15% SDS-PAGE and transferred to nitrocellulose membrane for all the proteins except for LC3, where PVDF membranes were used for LC3 signal. The membrane was blocked in 5% skimmed milk-TBST solution for 1 hour at room temperature. The membrane was probed with primary antibodies at 4°C overnight. Antibodies to Akt, phosphor-Akt at Ser473 (p-Akt), mTOR, phosphor-mTOR at Ser2448 (p-mTOR), LC3A/B, p62, ribosomal protein S6, ribosomal protein phosphor-S6 at Ser235/236 (p-S6), and Na/K-ATPase were purchased from Cell Signaling Technology. The antibodies to glucose transporter 4 (GLUT4) (H-61) and EIF-5 were bought from Santa Cruz Biotechnology (CA, USA). EIF-5 was measured as an internal control of total protein. Na/K-ATPase was used as a loading control of plasma membrane protein. The membrane was washed for 5 min with TBS-T buffer for 5 times and then incubated with a horseradish peroxide-conjugated secondary antibody at room temperature for 1 h. After washing, the membrane was developed with ECL Reagent (Millipore, USA) and exposed to Kodak XBT-1 film. Protein expression level was quantified with Image J (NIH) software.

Cytoplasmic and nuclear proteins of neonatal rat and adult mouse ventricles were extracted using nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL) according to the manufacturer's instructions. The protein concentration of the myocardial samples was determined using a protein concentration assay (Bio-Rad Laboratories, Hercules, CA). The extracted proteins were boiled in protein buffer for 5 min, separated by 12% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was incubated at room temperature for 1 h in TBS-T (Tris-buffered saline containing 0.1% Tween 20) containing 5% skimmed milk to block nonspecific binding sites. The membrane was then incubated overnight at 4°C with the primary antibodies. The membrane was washed for 5 min with TBS-T buffer three times and then incubated with a horseradish peroxide-conjugated secondary antibody at room temperature for 1 h. Finally, the membrane was developed using ECL reagent (Vigorous Biotechnology, Beijing, China) and exposed to Kodak XBT-1 film.