Anti p62 5114

Whole cell extracts were prepared using M-PER^® (ThermoFisher, Waltham, MA, USA) supplemented with 1× protease inhibitor cocktail (Complete EDTA-free, Roche, Basel, Switzerland), according to the manufacturer’s instructions. Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Science, Chicago, IL, USA). Membranes were incubated with selected primary antibodies: anti-caspase 7 (9494S), anti-caspase 9 (9502S), anti-caspase 8 (9746), anti-Mcl-1 (4572S), anti-LC3B (2775), and anti-p62 (5114) from Cell Signaling (Danvers, MA, USA), anti-PARP-1 (C2-10; sc-53643) from Santa Cruz Biotechnology (Dallas, TX, USA), anti-Bcl-xL (610212) from BD Pharmingen (San Jose, CA, USA), and anti-β-actin (5441) from Sigma Aldrich (St. Louis, MO, USA).
Luminescence signals were detected with the enhanced luminol-based chemiluminescent (ECL) Plus Western Blotting Detection Systems (GE Healthcare Life Science, Chicago, IL, USA). Bands were acquired by using an Amersham Imager 600 (GE Healthcare Life Science, Chicago, IL, USA) and quantified by ImageJ 1.52a software (U.S. National Institute of Health, Bethesda, MD, USA).

THP-1 cells coincubated with different RBCs were collected. Wild-type, shNC, and low-NLRP3 expression THP-1 cells were triggered with 800 nmol/L of rapamycin to create the autophagy positive control. THP-1 cells were lysed in a radio immunoprecipitation assay (RIPA) buffer (C500005, Sangon Biotech). The expression of autophagy markers on THP-1 was detected using immunoblotting with anti-p62 (5114) and anti-LC3 (2775, Cell Signaling Technology). Anti-NLRP3 (AG-20B-0014-C100, AdipoGen) and antigasdermin D (GSDMD) (HA601046, Hangzhou HuaAn Biotechnology Co., Ltd.) were applied to determine the activation of NLRP3 inflammasome. GAPDH (TA802519, OriGene Technologies) was used as internal control. Each blot was duplicated three times. BandScan software was utilized after development to analyze and record the gray value of the strip. Relative amounts of p62/GAPDH, LC3II/GAPDH, and LC3II/LC3I were adopted to estimate the autophagy situation of THP-1 cells, while those of NLRP3/GAPDH and GSDMD/GAPDH were used to evaluate the activation of NLRP3 inflammasome.

Cell pellets were homogenized in RIPA buffer (Sigma-Aldrich) supplemented with 1% protease and phosphatase inhibitors (Sigma-Aldrich). Samples were kept on ice for 30 min and afterwards centrifuged at 16,000 g for 20 min at 4°C, followed by protein quantification by the bicinchoninic acid assay (Pierce, Thermo Fisher Scientific). Homogenized samples were separated by SDS-PAGE, and transferred to nitrocellulose membranes (Amersham Pharmacia). The membranes were then probed using the following antibodies: anti-Lc3 (3868, Cell Signaling Technology; 1:1000), anti-p62 (5114, Cell Signaling Technology; 1:200), anti-beclin1 (sc-11427, Santa Cruz Biotechnology; 1:500) and anti-α-tubulin (T5168, Sigma-Aldrich; 1:1000). The specificity of antibodies was assessed using positive controls. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) and revealed using a Novex™ ECL Chemiluminescent Substrate Reagent kit (Invitrogen). For the reaction with the endogenous control, membranes were stripped with a stripping buffer (Thermo Fisher Scientific) and re-probed for α-tubulin analysis. Bands were quantified by densitometry using ImageJ.