Ab96899

We inoculated HK-2 cells (3 × 10⁴ cells/mL) into a 6-well plate with cover glass until the cell confluence reached about 70%. After the cells received different treatments, we fixed the cells with a 4% paraformaldehyde fixation solution (P0099, Beyotime, China) and then rinsed them thoroughly with phosphate buffer (PBS, SH30256.01, Hyclone, USA) for 3 times. After permeating the cell membrane with 0.5% Triton X-100 (T109027, Aladdin, China) in each hole, we sealed the cells with 3% BSA blocking solution (B265993, Aladdin, China) for 30 min. They were then reacted with anti-Caspase-3 antibody (1:500, ab32351, abcam, UK) at 4 °C overnight. After the cells in each well were reacted with goat anti-rabbit IgG H&L (DyLight® 488, 1:500, ab96899, abcam, UK), they were reacted with DAPI (ab104139, abcam, UK). In the end, after mounting, the cover glass was placed under the inverted fluorescence microscope (Ts2-FC, Nikon, Japan) to observe the results.

For IF staining, sections were deparaffinized. After antigen blocking and retrieval, the sections were incubated with anti-IL-1RA antibodies (ab124962, Abcam, Cambridge, UK) at 4 °C overnight. They were rewarmed at RT for 30 min and thoroughly rinsed with PBS. Secondary Dylight 488 conjugated goat anti-rabbit antibodies (ab96899, Abcam, Cambridge, UK) were added and incubated at 37 °C for 1 h and stained with a fluorescent mounting medium with DAPI. A confocal imaging system (TCS-SP8, Leica, Germany) was used to analyze all images.

27-Hydroxycholesterol was provided by American Santa Cruz, and 27-OHC (10 mg) was completely dissolved in a suitable amount of ethanol, then divided equally in centrifuge tubes, with nitrogen gas blowing dry, and finally preserved at −80°C. The 27-OHC working solution contains 0.16% ethanol (v/v). Filipin III was from Cayman Chemical (#70440, Ann Arbor, MI, United States). Primary antibodies for Western blot included mouse anti-ABCA1 (Abcam, ab66217), rabbit anti-ABCG1 (Abcam, ab52617), mouse anti-Caveolin-1 (Abcam, ab17052), rabbit anti-Apolipoprotein E (Abcam, ab52607), mouse anti-LDLR (Millipore, MABS26), rabbit anti-ACAT1 (Abcam, ab168342), rabbit anti-CYP46A1 (Sigma–Aldrich, SAB2100523), mouse anti-KDEL (Santa Cruz, sc-58774), rabbit anti-flotillin (Abcam, ab41927), rabbit anti-SR-B1 (Abcam, 52629), mouse anti- N Cadherin (Abcam, ab98952), mouse anti-Aβ (Abcam, ab126649), and rabbit anti-LRP1 (Abcam, ab92544). Goat anti-mouse (#14709) and rabbit (#7074) biotinylated secondary antibodies were from Cell Signaling Technology. Antibodies for immunofluorescence included anti-CYP46A1 (Abcam, ab82814), goat anti-mouse (Abcam, ab150120), and goat anti-rabbit (Abcam, ab96899).

Lung tissues and RAW264.7 cells were lysed in RIPA lysis buffer (P0013B,Beyotime) supplemented with protease inhibitor (ST505, Beyotime) and phosphatase inhibitor (P1050, Beyotime), and the protein concentration in each sample was determined using the BCA assay. Samples were denatured, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The non-specific membrane binding sites were blocked with 5% bovine serum albumin (BSA) for 1 h. Membranes were then incubated at 4°C overnight with primary antibodies (all diluted 1:1000) against the following proteins: IL-17 (ab79056, Abcam), MCP-1 (catalog no. 2029, CST),p38 MAPK (catalog no. 9212, CST), and phosphorylated (p)-p38 MAPK (catalog no. 4511, CST). Blots were washed several times with PBS buffer, then incubated for 1 h with goat anti-rabbit IgG secondary antibody (diluted 1:15000; ab96899, Abcam). The bands were visualized using a fluorescent scanner. As an internal reference, GAPDH was immunostained using an antibody (1:1000; catalog no. 5174, CST).

Tissues were lysed using lysis buffer (Beyotime, Beijing, China). Protein concentrations were measured with a BCA kit (#ab102536, Abcam, UK). 40 µg protein was separated using SDS-PAGE, and transferred to a PVDF membrane (Millipore). The proteins were blocked with TBST buffer (#ab64202, abcam, UK) for 2 h. After washing twice, proteins were incubated with primary antibodies at 4°C overnight, and then incubated with secondary antibodies for 2 h after washing. Proteins were detected with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, USA), and bands was analyzed with ImageJ software. The antibodies used are: Rabbit monoclonal to SOCS1 (ab280886, 1:1000, abcam, UK), Rabbit monoclonal to GAPDH (ab181602, 1:1000, abcam, UK), Goat Anti-Rabbit IgG (ab96899, 1:2000, abcam, UK). GAPDH was used as a housekeeping control gene in the experiments as described previously [26 (link)].

OCCM-30 cells were cultured overnight on sterile Falcon™ Chambered Cell Culture Slides (354108, Fisher Scientific) and further fixed with 4% paraformaldehyde (30525-89-4, Sigma-Aldrich) for 10 min at room temperature. Cells were permeabilized with 0.5% Triton™ X-100 Surfact-Amps™ Detergent Solution (28313, Thermo-Fisher) for 20 min. Then, cells were incubated in blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA (071M8410, Sigma) and 0.1% Tween-20 (P1379, Sigma-Aldrich) for 30 min at room temperature and further incubated with primary antibodies AdipoR1 (ab70362, Abcam) (dilution 1:250) or AdipoR2 (ab77612, Abcam) (dilution 1:250) at 4°C overnight. The secondary antibodies DyLight 488 polyclonal goat anti-rabbit (ab96899, Abcam) (dilution 1:500) or donkey anti-goat Alexa Fluor 647 (ab150131, Abcam) (dilution 1:500) conjugated to fluorescein isothiocyanate were used. After washing with 1× phosphate-buffered saline (PBS) (10010023, Thermo-Fisher), samples were mounted using a fluorescent Mounting Medium with DAPI (ab104139, Abcam). Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and photographed.

The total proteins isolated from cells were measured using BCA kits and lysis buffer (RIPA, Cell Signaling) (Beyotime, China). All the protein samples (30 μg) were split up using an SDS polyacrylamide gel and then conveyed to polyvinylidene difluoride (PVDF) membranes (Invitrogen). After blocking at room temperature with 5% skim milk for 1 h in PBS and then at 4°C overnight, the PVDF membranes were protected with primary antibodies anti-RhoB (ab155149; 1 : 1000; Abcam) and anti-GAPDH (ab9485; 1 : 1000; Abcam). Membranes were washed, and then, a goat anti-rabbit secondary antibody preincubated for an hour and labeled with horseradish peroxidase (ab96899; 1 : 1000; Abcam) was used and then rinsed with PBS. ECL detection reagent was used to visualize the protein bands (Pierce, Rockford, IL, United States). ImageJ software was used to quantify western blot densitometry bands.