Ab62557

Protein samples were extracted from myocardial tissue and H9C2 cells. Protein concentrations were determined using the BCA method to ensure consistent loading of each sample. Proteins were separated using SDS-PAGE, and then transferred to 0.45-μm PVDF membranes. After blocking with skim milk for 1 hour, membranes were incubated with antibodies against Bcl-2 (ab32124), Bax (ab53154), p53 (ab32389), LC3B(ab63817), SQSTM1/62 (ab91526), ATG5 (ab53154), Beclin1 (ab62557), or β-actin (ab8226) (antibodies purchased from Abcam Biotechnology, USA) at 4 °C overnight. After labeling with secondary antibodies, the target protein was observed using enhanced chemical luminescence.

Primary HESCs, human peritoneal macrophages, THP-1 cells, and HESCs after multiple treatments were fixed in ice-cold methanol for 15 min, washed three times with 4°C PBS, incubated for 15 min in 0.25% Triton X-100 diluted in PBS, immersed in PBS twice for 3 min, and blocked with 1% BSA in PBS for 30 min at about 26°C. The primary HESCs and HESCs were incubated with anti-Beclin1 (1:100; ab62557, Abcam), anti-LC3B (1:100; ab51520, Abcam), SQSTM1/p62 (1:500; ab109012, Abcam), and ULK1 (1:200; ab203207, Abcam). Human peritoneal macrophages and THP-1 cells were incubated with anti-MST1 (1:400; ab51134, Abcam) and anti-p38-MAPK (1:200; ab170099, Abcam). The incubation time of each antibody varied from 1 h to 24 h at 4°C. All antibodies were diluted in 1% BSA in PBS. After cells were immersed in PBS twice for 3 min, they were incubated with the anti-mouse/rabbit secondary antibody for 30 min at about 26°C. Anti-quench nuclear staining and mounting with DAPI (ab104139, Abcam, USA) were performed. Images were captured with a confocal microscope (LSM880 Airy, Zeiss) and processed with the Zen software (Zeiss).

The nasal tissues in AR group and NR group were prepared into a homogenate and centrifuged at 2,000 r/min for 20min, and the supernatant was collected. The protein concentration was measured using BCA kit (Solarbio, Beijing, China), 40μl protein sample and 10% SDS gel buffer were mixed by 1:1 and heated 5min at 95°C for protein denaturation. Then, the mixture was transferred onto a polyvinylidene difluoride (PVDF) membrane at 80V (Merck, Darmstadt, Germany) for 30 min, and then the PVDF membrane was blocked with TBST solution containing 5% defatted milk powder at 4°C for 1h, added with rabbit anti-human FOS (1:1000, ab190289, Abcam), Bcl-2 (1:1000, ab196495, Abcam), Bax (1:1000, ab263897, Abcam), Beclin1 (1:1000, ab62557, Abcam), p62 (1:1000, ab155686, Abcam), and β-actin (1:2000, orb178392, Biorbyt, Cambridge, UK) polyclonal antibodies which were diluted with TBST solution containing 3% FBS protein, and then incubated overnight at 4°C. After re-warming, the PVDF membrane was incubated with horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1:1000, ABIN101988, antibodies-online, Aachen, Germany) for 1h, washed, and developed with ECL luminescent substrate for 3-5min. The protein expression level was normalized using β-actin, and the gray scan and quantification were performed with Image J(NIH) software.