Ab221794

Five-µm-thick sections of the formalin-fixed paraffin-embedded hepatic tissues were stained for Beclin-1 and LC3-II. Heat-mediated antigen retrieval was performed using citrate buffer, pH 6.0, and microwaved for 20 min. The sections were then incubated with the rabbit polyclonal anti-Beclin-1 primary antibody (ab62557) (Abcam, Cambridge, UK) at 1 µg/mL dilution and the rabbit monoclonal recombinant anti-LC3B primary antibody [EPR18709] (ab221794) (Abcam, Cambridge, UK) at 0.1 μg/mL dilution following the avidin-biotin-peroxidase complex technique [52 (link)]. The substance 3,3′-Diaminobenzidine was used as chromogen and hematoxylin as counterstain. Image J’s color deconvolution plugins were then used to quantify the immunoexpression of Beclin-1 and LC3-II in liver tissues in ten high-power microscopic fields per marker per rat [53 (link)].

The cells were harvested, lysed using ice-cold NP-40 lysis buffer (Cell Signaling Technology), centrifuged and the supernatant collected. Equal amounts of the supernatant protein (50 μg) were loaded onto an SDS-PAGE gel. The samples were electrophoresed and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA). WB analysis of cell lysates [28 (link),29 (link)] was carried out using the following primary antibodies: SOCS5 (ab244384, 1:2000), Bcl-2 (ab182858, 1;1000), p62 (ab280086, 1:2000), LC3B (ab221794, 1:2000), Beclin 1 (ab207612, 1:2000), ATG7 (ab52472, 1:2000), Bcl-2 (phospho S70, ab218123, 1:2000), cleaved Caspase-3 (ab32042, 1:3000), cleaved Caspase-9 (ab2324, 1:3000), and GAPDH (ab9485, 1:1000), which were purchased from Abcam. After the membranes were washed three times for 10 min with Tween-Tris-buffered saline buffer, they were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 120 min. The positive signal for the target protein was analyzed using a Tanon 2500 image analyzer (Shanghai, China). Densitometry of specific blotted bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/), and the intensity values were normalized against the GAPDH loading control.

Primary antibodies targeting LC3B (1:1000; Abcam, ab221794, 14, 16 kDa), Atg5 (1:1000; Abcam, ab108327, 55 kDa), Atg7 (1:1000; Abcam, ab133528, 77 kDa), p62 (1:1000; Abcam, ab109012, 62 kDa), ALKBH5 (1:1000; Abcam, ab195377, 44 kDa), p-GSK3β (Ser9, 1:1000; Abcam, ab107166, 47 kDa), GSK3β (1:1000; Abcam, ab280376, 46 kDa), p-mTOR (S2448, 1:1000; Abcam, ab109268, 289 kDa), mTOR (1:1000; Abcam, ab134903, 289 kDa), and Beclin-1 (1:1000; Abcam, ab207612, 52 kDa) were used. Horseradish peroxidase (HRP)-linked anti-mouse IgG and anti-rabbit IgG (1:5000; Protein Tech Group, SA00001-2) were used, respectively. Data were normalized to β-actin expression. Immunoreactive bands were quantified by densitometry and computer-assisted image analysis.