Anti lc3b antibody

Western blotting was performed to evaluate the autophagy markers. Briefly, cell lysates were prepared according to the following protocol. First, cells were incubated in RIPA buffer [Tris-HCl (pH 7.4), NaCl (150 mM), NP40 (1%), Na-deoxycholate (0.25%), EDTA (1 mM), dithiothreitol (1 mM), and phenylmethylsulfonyl fluoride (1 mM)] with a freshly added protease inhibitor cocktail (Sigma Aldrich). Thereafter, the cell lysates were centrifuged at 12.000 rpm for 10 min and the resulting supernatants were collected for western blot analysis. The protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), and 30 μg of protein per sample was loaded onto a pre-cast SDS-PAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific). The proteins were then transferred to PVDF membranes which were incubated overnight with a rabbit polyclonal anti-LC3B antibody (Abcam, Cambridge, UK) while a mouse anti-GAPDH antibody (Thermo Fisher) was used as a loading control. After washing, the membranes were incubated with fluorescently labeled anti-mouse or anti-rabbit secondary antibodies obtained from LI-COR Biosciences (Lincoln, NE, USA). The blots were scanned and imaged using the LI-COR Biosciences Odyssey^® imaging system.

The spleen samples were lysed in 500 $\mathsf{\mu }$ L RIPA lysis buffer supplemented with 1% Phenylmethanesulfonyl fluoride. Following centrifugation at 13,000× g for 15 min at 4 ${}^{\circ }$ C, the concentration of the total protein was quantified using a BCA assay kit (Thermo Scientific, Rockford, IL, USA). The lysates were mixed with 5 × SDS loading buffer and heated at 99 ${}^{\circ }$ C for 10 min. Samples were then analyzed by SDS-PAGE essentially, as previous described [19 (link)]. The rabbit monoclonal anti-GAPDH antibody (Abcam, Cambridgeshire, Britain), rabbit polyclonal anti-LC3B antibody (Abcam, Cambridgeshire, Britain), rabbit anti-Beclin1 polyclonal antibody (Beyotime, Shanghai, China), rabbit anti-CD4 polyclonal antibody (Beyotime, Shanghai, China) and mouse anti-CD8 monoclonal antibody were used as primary antibodies, and HRP donkey anti-mouse IgG or HRP donkey anti-rabbit IgG (ABclonal, Wuhan, China) were served as the secondary antibody in the study. Protein bands in the membrane were detected using a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) after incubation of the membranes with Clarity Western ECL Blotting Substrate. Then, protein bands were analyzed by software Image-Pro Plus (Version 6.0.0.260), and protein levels were normalized to the amount of GAPDH.

Frozen samples of renal cortex obtained from db/+ mice and db/db mice treated with empagliflozin or empagliflozin–linagliptin were homogenized in protein lysis RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% of Triton X-100, 1% of sodium deoxycholate, 0.1% of SDS, and 1 mM EDTA). The homogenate was centrifuged twice at 14,000× g for 30 min at 4 °C to obtain the supernatant. Equal amounts of protein were analyzed by immunoblot assays using anti-LAMP1, anti-Caspase-3, anti-Bcl-2, anti-LC3B antibody (Abcam, Cambridge, UK, ab25245, ab13847, ab692 and ab48394, 1:1000) and β-actin (Abcam, Cambridge, UK, ab1801) as control. Intensities of individual bands were quantified by using ImageJ software (Madison, WI, USA).

The frozen RAA tissues were embedded in the optimum cutting temperature (OCT) compound and sectioned at −25°C in a cryostat. Sections (5 μm in thickness) were fixed with 4% polyformaldehyde for 15 minutes at room temperature. The sections were permeabilized with 0.1% TritonX-100 for 20 minutes followed by blocking with 10% goat serum for 1 hour at room temperature. Then, the sections were incubated with anti-Cox IV antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 200, anti-LAMP-1 antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 200, or anti-LC3B antibody (Abcam) diluted at 1 : 100 overnight at 4°C. Subsequently, the sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 500, or Alexa Fluor 594-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA) diluted at 1 : 500 for 1 hour at room temperature. DAPI (Solarbio Science & Technology, Beijing, China) was used to stain the cell nuclei. Finally, the fluorescence images were captured by using a laser scanning microscope and further analyzed by ImagePro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).

H9C2 cells were lysed with RIPA buffer mixed with proteinase inhibitor cocktail (Abcam, Britain) and PMSF (Sigma, USA). The cell extract was centrifuged at 12000 × g for 30 min at 4°C. Supernatant concentrations were measured using a BCA Protein Quantitation Kit (Thermo Fisher Scientific, USA). The concentration of the protein precipitate was proportional to the supernatant. After denaturation, both the supernatants and precipitates were separated via SDS-PAGE, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Membranes incubated in the corresponding antibodies and stored at 4°C overnight. Anti-LC3B antibody and anti-P62 antibody were purchased from Abcam (Britain). Anti-Ambra1 antibody was purchased from Cell Signaling Technology (USA). Anti-β-actin antibody was purchased from Solarbio (China). After washing with TBST (Solarbio, China), the secondary antibody (Abcam, Britain) was incubated. Finally, the membranes were visualized using the Gel Doc XR+ System (Bio-Rad, USA).