Rabbit anti tlr4

qPCR and Western blot were performed as previously reported 19 (link). qPCR was carried out using the Hairpin-it^TM microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and LightCycler 96 (Roche, Germany). U6 snRNA was used as an internal control for miR-92b, and the mRNA levels of PTEN, TNF-α and IL-6 were normalized to GAPDH. The 2^-ΔΔCt method was employed to calculate relative expression of each gene. For Western blot analysis, the protein blots were incubated with following antibodies: rabbit anti-TLR4 (1:1000, 7074, Cell Signaling Technology, USA), rabbit anti-phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (1:1000, 4764, Cell Signaling Technology, USA), rabbit anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), rabbit anti-Bax (1:200, bs-0127R, Bioss, China), rabbit anti-Bcl-2 (1:200, bs-4563R, Bioss, China), rabbit anti-phospho-PI3K (1:200, bs-3332R, Bioss, China), rabbit anti-PI3K (1:1000, ab227204, Abcam, USA), rabbit anti-phospho-AKT (1:1000, ab8932, Abcam, USA), rabbit anti-AKT (1:500, GB111114, Servicebio, China), rabbit anti-β-catenin (1:5000, ab196204, Abcam, USA). The optical density (OD) value of each protein band was quantified using the Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics Inc., USA) and normalized to β-actin.

Thioglycolate-elicited peritoneal macrophages were seeded in 10% FBS containing DMEM and incubated overnight. Unadhered cells were washed off, and 1% bovine serum albumin (BSA) containing DMEM was added to the plate and incubated for 3 h before ginkgetin treatment. To determine expression of CD36, TRPV4, TLR2, TLR4, TLR6, and GAPDH proteins whole cell lysates were prepared from cells treated overnight with ginkgetin (1 and 5 µM) or vehicle in the presence or absence of oxLDL (25 µg/ml). To determine the expression levels of LPS-triggered p-JNK and JNK, cells were pretreated with ginkgetin (5 and 10 µM) for 3 h and then stimulated with E. coli LPS for 30 min. Whole cell lysates were prepared from twice-washed cells (ice cold PBS) using RIPA buffer with added protease-inhibitor cocktail (Thermo Scientific, MA). Blots were probed with rabbit anti-TRPV4 (Alomone Lab, Jerusalem, Israel), anti-mouse CD36 (R&D, Minneapolis, MN), rabbit anti-TLR4, rabbit anti-TLR6, rabbit anti-JNK, and rabbit anti-p-JNK primary antibodies (Cell Signaling, Danvers, MA) followed by anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (R&D, Minneapolis, MN). Blots were stripped and re-probed with anti-GAPDH IgG (1:2000; Santa Cruz, Dallas, TX) for loading control.

Western blot procedures were performed as previously described [16 ]. The primary antibodies were rabbit anti-calcium/calmodulin-dependent protein kinase II (CaMKII) (1 : 1000, ab52476, Abcam, Cambridge, United Kingdom), rabbit anti-phospho- (P-) CaMKII (1 : 1000, ab5683, Abcam), rabbit anti-AKT (1 : 1000, #4685, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-P-AKT (1 : 1000, #4060, Cell Signaling Technology), rabbit anti-P-ERK1/2 (1 : 1000, #4370, Cell Signaling Technology), rabbit anti-ERK1/2 (1 : 1000, #4695, Cell Signaling Technology), rabbit anti-P-p38 (1 : 1000, #9215, Cell Signaling Technology), rabbit anti-p38 (1 : 1000, #9212, Cell Signaling Technology), rabbit anti-P-SAPK/JNK (1 : 1000, #4668, Cell Signaling Technology), rabbit anti-SAPK/JNK (1 : 1000, #9258, Cell Signaling Technology), rabbit anti-TLR4 (1 : 1000, #14358, Cell Signaling Technology), rabbit anti-NF-κB p65 (1 : 1000, #8242, Cell Signaling Technology), rabbit anti-P-NF-κB p65 (1 : 1000, #3033, Cell Signaling Technology), and rabbit anti-β-actin (1 : 2000, #4970, Cell Signaling Technology). Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (1 : 5000, A8275, Sigma-Aldrich) was used as a secondary antibody.

Western blotting was performed as described in our previously published study (Huang et al. 2020 ). The protein concentration of each sample was measured using a BCA Protein Assay Kit, and then the samples were heated for 5 min at 99 °C. Next, 30 µg of protein was loaded in each well of a 10% polyacrylamide gel, and then the proteins on the gel were transferred to a polyvinylidene fluoride membrane. The PVDF membrane was incubated in 5% skimmed milk for 1 h, washed and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-TLR4 (Cell Signaling Technology (CST); 1:1000), rabbit anti-phospho-p44/42 MAPK (Erk1/2) (CST; 1:2000), rabbit anti-p44/42 MAPK (Erk1/2) (CST; 1:2000), rabbit anti-phospho-NF-κB p65 (CST; 1:1000), rabbit anti-NF-κB p65 (CST; 1:1000), rabbit anti-Iba1 (FUJIFILM Wako Chemicals; 1:500), mouse anti-GFAP (CST; 1:2000) and rabbit anti-GAPDH (CST; 1:5000) followed by peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:3000; Jackson ImmunoResearch) and a peroxidase-conjugated donkey anti-goat secondary antibody (1:2000; Jackson ImmunoResearch). Finally, the proteins were visualized using Western peroxide reagent and Clarity Western ECL Substrate (Bio-Rad) and the ChemiDoc XRS system (Bio-Rad) with Image Lab software. NIH ImageJ software was used to quantify the intensity of the bands by measuring the optical density.