P62 antibody

Doxorubicin was purchased from Cell Signaling Technology (Danvers, MA, USA). LC3B antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA); caspase-3 antibodies were purchased from Santa Cruz (Dallas, TX, USA) and Cell Signaling Technology (Danvers, MA, USA); p62 antibody and horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, USA); CyclinG1 antibody was purchased from Proteintech Group (Chicago, IL, USA). Unless otherwise specified, all other chemicals were purchased from Sigma (St. Louis, MO, USA) or Solarbio (Beijing, China).

After intervention, the cells were collected and subjected to protein extraction. After electrophoresis and membrane transfer, incubation with primary antibodies against Mst1 (3682 S; CST), phospho-Mst1 (Thr183)/MST2 (Thr180) (49,332 S; CST), Beclin-1 (3738 S; CST), p62 antibody (23,214 S; Cell Signalling), LC3-I and LC3-II antibody (2775 S; Cell Signalling) was performed. Primary antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA). After that, the membrane was incubated with corresponding HRP-conjugated secondary antibodies (goat against rabbit, ab205718; and goat against mouse, ab205719; Abcam). Color development was performed and detected with the ChemiScope mini chemiluminescence instrument. β-actin was used as internal control.

For HSP70 and HSP27 analysis proteins were separated on pre-cast 10% criterion gels (BioRad) and membranes were incubated with HSP70 antibody (Enzo) or HSP27 antibody (Cell Signaling), and GAPDH antibody (Cell signaling) to correct for loading differences. For LC3B-I and LC3B-II analysis proteins were separated on pre-cast 8–16% gradient TGX gels (BioRad) and membranes were incubated with LC3B-I/II antibody. Analysis of p62 expression was performed by separating proteins on a 12% acrylamide gel and the membrane was cut in two parts. The upper part was incubated with p62 antibody (Cell signaling) and the lower part with GAPDH antibody. Data from different blots was combined by loading the same sample on each gel and normalizing all for this sample. Images were captured with AI600 (GE Healthcare Life Sciences) and analyzed with ImageQuantTL software (GE Healthcare Life Sciences). Raw blot images of the representative images can be seen as Supplementary Material online.

Spinetoram (CAS No: 187166-40-1, purity > 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin–streptomycin were obtained from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Gibco (Norristown, PA, USA). Phosphate buffered saline (PBS) was obtained from Servicebio (Wuhan, China).
The malondialdehyde (MDA) content assay kit (D799762-0100), reduced glutathione (GSH) content assay kit (D799614-0100), catalase (CAT) activity assay kit (D799598-9100), and superoxide dismutase (SOD) activity assay kit (D799594-0100) were bought from Sangon Biotech (Beijing, China). The LC3 antibody, Beclin1 antibody, p62 antibody, p-mTOR antibody, p-AMPK antibody, GAPDH antibody, and γH2AX antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). The Alexa fluor 488-conjugated antibody were purchased from Sangon Biotech (Shanghai, China).

Total protein was isolated from murine BMDMs and colonic tissues using a standard extraction reagent supplemented with protease inhibitor (Kangchen; Shanghai, China). The concentration of extracted protein was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins were then separated using SDS-PAGE and electro-transferred to nitrocellulose membranes as previous described [50 (link)]. Immunoblotting was performed using a Beclin-1 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), an LC3 antibody (1:500; Novus Biologicals, Littleton, CO, USA), and a p62 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA) for 1 h at 25 °C. Finally, images of the blots were obtained using an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA).

Protein lysates were extracted from brain tissues or culture cells by RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Sigma, USA). Protein concentration was measured by BCA kit (Beyotime, China). A total of 20 μg protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Biorad, USA). Then, the membranes were blocked by 5% fat-free milk for 1 h at room temperature, incubated with first antibodies at 4 °C overnight and corresponding second antibodies for 1 h at room temperature. Then antibodies used in our study were: iNOS Rabbit mAb (CST#13120, 1: 1000), Arginase-1 Rabbit mAb (CST#93668, 1: 1000), GAPDH Rabbit mAb (CST#2118, 1: 1000), LC3A/B Rabbit mAb (CST#12741, 1: 1000), p62 Antibody (CST#5114, 1: 1000), Phospho-Akt (Ser473) Antibody (CST#9271, 1: 1000), Akt Antibody (CST#9272, 1: 1000), Phospho-mTOR (Ser2448) Antibody (CST#2971, 1: 1000), mTOR Rabbit mAb (CST#2983, 1: 1000), pro-IL-1β Rabbit mAb (CST#31202, 1: 1000), Cleaved-IL-1β Rabbit mAb (CST#63124, 1: 1000), pro-Caspase-3 Antibody (CST#9662, 1: 1000), Cleaved Caspase-3 Antibody (CST#9661, 1: 1000), Phospho-GSK-3α/β (Ser21/9) Antibody (CST#9331, 1: 1000), GSK-3α/β Rabbit mAb (CST#5676, 1: 1000) and NLRP3 Rabbit mAb (CST#15101, 1: 1000) were all obtained from Cell Signaling Technology (USA).