Arg51850

Total protein was obtained from snap-frozen hearts or cells using a protein extraction kit (Keygenbio, KGP250) and a 1.5 mL tube (Guangzhou Jet Bio-Filtration Co., Ltd.). Thirty micrograms of protein was separated by SDS–PAGE and then transferred onto PVDF membranes. The membranes were incubated at 4°C overnight with conjugated antibodies (Sino Biological Inc., 1 : 2000). The immunoblotting bands were detected by chemiluminescence. The following antibodies were used: anti-VEGF-C (22601-1-AP, Proteintech), anti-VEGFR-3 (ab273148, Abcam), anti-p-p38 (ARG51850, Arigo), anti-p38 (ARG55258, Arigo), anti-VE-cadherin (ab205336, Abcam), anti-p-ERK1/2 (4370, CST), anti-ERK1/2 (4695S, CST), anti-p-AKT (9271S, CST), anti-AKT (9272, CST), anticalcineurin A (CaNA) (2614, CST), anti-p-STAT3 (9145, CST), anti-STAT3 (9139, CST), anti-TGF-β1 (3711, CST), anti-p-Smad2/3 (882S, CST), anti-Smad2/3 (8685, CST), anti-NOX2 (19013-1-AP, Proteintech), anti-NOX4 (14347-1-AP, Proteintech), anti-p-P65 (3033, CST), anti-P65 (4764, CST), anti-β2i (13635, CST), anti-β5i (ARG41022, Arigo) anti-MKP5 (374276, Santa), and anti-GAPDH (5174, CST). ImageJ software was used for densitometry analysis.

Shikonin was purchased from ChemFaces (Wuhan, China). Thiazolyl blue tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), NAC, and other chemicals were obtained from Sigma-Aldrich (St. Louis, MI, USA). Anti-poly-ADP ribose polymerase was purchased from Cell Signaling Technology (No. 9542S, Beverly, MA, USA). Antibodies against ERK1/2 (ARG62350), phosphorylated ERK1/2 (ARG52277), and phosphorylated p38 (ARG51850) were obtained from Arigo Biolaboratories (Hsinchu City, Taiwan). Caspase 3 antibody (GTX110543) was purchased from GeneTex, Inc. (Hsinchu City, Taiwan). Light chain-3 (LC3)-II (NB 100-2220) antibody was obtained from Novus Biologicals (Centennial, CO, USA). Actin (6008-1-IG), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-Ig), and p38 (66234-1-Ig) were obtained from Proteintech Group, Inc. (Rosemont, IL, USA). Bcl-2 (TGABF-188784-25) was obtained from TrustGene Biotech Company (Taichung, Taiwan).

Total PBMC protein was extracted from cells using cell lysis buffer containing PMSF (Beyotime). The samples were separated by 12.5% SDS-polyacrylamide gel electrophoresis, followed by transferring to the PVDF membrane. After blocking in PBS containing 5% bovine serum albumin, the membrane was incubated with primary Rab31 antibody (1:1,000; 16182-1-AP, Proteintech), NLRP3 antibody (1:1,000; 15101,Cell Signaling Technology), p-P38 antibody (1:1,000; ARG51850, Arigo), or beta-actin antibody (1:5,000; 380624, Zen BioScience) at 4°C overnight, followed by incubation with a peroxidase-linked secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, 1:5,000; 7074S, Cell Signaling Technology; or Anti-mouse IgG, HRP-linked Antibody, 1:2,000; 7076S, Cell Signaling Technology) at room temperature for 1 h. The signals were detected by X-ray film after incubating with Western blotting Luminol reagent (GE Health care). All images were analyzed with ImageJ software.