P ikkβ

The wild-type HSV-2 strain 186 and the Us2-deficient (YY2) was obtained from the State Key Laboratory of Virology (Wuhan University) and both viruses were generated as described previously^{25 (link)}. The HSV-2 Us region expression plasmids were kind gifts of Dr. Yefu Wang, Wuhan University. NF-κB-luc plasmid were kind gifts of Dr. Qi Zhang, Wuhan University. Viruses were propagated on Vero cells and stored in aliquots at −80 °C until use. Viral titers were measured in Vero cells and expressed as plaque forming units (PFU)/ml and used for infection studies at a multiplicity of infection (MOI) of 1. UV-inactivated HSV-2 was obtained by exposure to UV irradiation for 20 min.
Antibody against β-tubulin and Lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human and mice antibodies against TAK1, p-TAK1, IKKβ, p-IKKβ, IκB, and p-IκB were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against HSV-2 Us2 was purchased from WuXi AppTec (Shanghai, China). Antibody against Flag and HA were purchased from Sigma (St. Louis, MO, USA). All culture plasticware was obtained from Corning (Corning, NY, USA).

Oxidized low‐density lipoproteins (oxLDL) and DiI‐labeled oxLDL (DiI‐oxLDL) were purchased from Peking Union‐Biology (Beijing, China). Antibodies against GAPDH (#5174, 1:1,000), β‐actin (#3700, 1:1,000), p‐IKKβ (#2697, 1:1,000), IKKβ (#8943, 1:1,000), p‐p65 (#3033, 1:1,000 for western blotting and 1:200 for immunofluorescence staining), NF‐κB p65 (#8242, 1:1,000), and IκBα (#9242, 1:1,000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against DCLK1 (#ab31704, 1:1,000 for western blotting and 1:200 for immunofluorescence staining), F4/80 (#ab6640, 1:200), CD31 (#ab9498, 1:200), α‐SMA (#ab7817, 1:200), iNOS (#ab178945, 1:200), and Lamin B1 (#ab133741, 1:1,000) were purchased from Abcam (Cambridge, UK). Antibodies against Ly6G (#sc‐53515, 1:100) and Ly6C (#sc‐271811, 1:100) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against Flag (#20543‐1‐AP, 1:1,000) was purchased from Proteintech (Wuhan, China). DCLK1‐IN‐1 (BD01203503) was purchased from Bidepharm (Shanghai, China).

Proteins (30 μg) were loaded on a precast gel in equal amounts to perform electrophoresis. After the electrophoresis, proteins on the gel were transferred to a nitrocellulose membrane which was blocked by incubating for 1 hour with 5% milk at room temperature. Primary antibodies for fibronectin(Cat: 26836), N‐cadherin(Cat: 13116), E‐cadherin(Cat: 14472), p‐p65 (Ser536)(Cat: 3033), α‐Tubulin(Cat:2125), Lamin A/C(Cat:4777), and β‐Actin(Cat: 4970) p‐IKKβ (Ser177)(Cat:2078), IKKβ(Cat:8943), p‐IκBα (Ser32)(Cat:5210), and IκBα(Cat:9242) (Cell Signalling Technology, Danvers, MA), p65(Cat:PA5‐16545), AKIP1(Cat:PA5‐106533), p‐Akt (Ser473)(Cat: OMA1‐03061), Akt(Cat:44‐609G), (Thermofisher, Massachusetts) were used during subsequent membrane incubation for 12 hours at 4°C. Following rinsing extensively with Tris‐buffered saline‐Tween 20, membranes were blotted with secondary antibodies conjugated to horseradish peroxidase (HRP) (Bio‐Rad) for 1 hour, and chemiluminescence kit was used to detect the signal intensity, which was quantified with densitometry using NIH ImageJ software (ImageJ, RRID: SCR_003070) and a Xerox scanner.

Total protein was isolated from renal cortical tissues in protein lysis buffer (Beyotime, Jiangsu, China). Cytoplasmic and nuclear proteins were extracted from renal cortical tissues using a Nuclear/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA) according to the manufacturer's instructions. The protein concentrations were determined using a bicinchoninic acid assay kit (Beyotime). Protein from each sample (50 μg) was separated by 10% SDS‐acrylamide gel and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with the following primary antibodies at 4 °C overnight: p65, p50, p‐IκBα, IκBα, and p‐IKKβ (dilution 1 : 1000; Cell Signaling Technology); Lamin B and β‐actin (diluted 1 : 2000) (Santa Cruz). On the second day, the membranes were washed and incubated with appropriate horseradish peroxidase‐conjugated secondary antibodies (anti‐rabbit or anti goat; Santa Cruz) at a 1 : 4000 dilution. Blots were visualized with enhanced chemiluminescence (Thermo, Rockford, IL, USA) and the relative density was quantitated using the image‐pro plus software (Media Cybernetics, Rockville, MD, USA).