Mouse anti ubiquitin

The primary antibodies used in this study were affinity-purified rabbit polyclonal anti-InlC (R134) (16 (link)), mouse antiubiquitin (catalog number 3936; Cell Signaling Technology), rabbit anti-SUMO1 (catalog number 4930; Cell Signaling Technology), mouse anti-ISG15 (F-9, catalog number sc166755; Santa Cruz Biotechnology), mouse anti-HA (6E2, catalog number 2367; Cell Signaling Technology), rabbit anti-HA (C29F4; Cell Signaling Technology), mouse antiactin (AC-15; Sigma-Aldrich), rabbit anti-S100A9 (catalog number pab0423-P; Covalab), and mouse anti-NF-κB-p65 (F6; Santa Cruz).

Proteins from ipsilateral cortical tissue were extracted using RIPA buffer, equalized, and loaded onto 5–20% gradient gels for SDS-PAGE (Bio-Rad, Hercules, CA, USA). Proteins were transferred onto nitrocellulose membranes and then blocked overnight in 5% milk in 1× TBS containing 0.05% Tween-20 (TBS-T). The membrane was incubated in rabbit anti-IBA1 (1:1,000; BD Transduction Laboratories), mouse anti-caspase 12 (1:1,000; Cell Signaling Technology), mouse anti-ubiquitin (1:1,000; Cell Signaling Technology), mouse anti-phospho-H2AX (1:1,000; Cell Signaling Technology), and rabbit anti-GAPDH (1:2,000; Sigma) overnight at 4°C, then washed three times in TBS-T and incubated in appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Membranes were washed three times in TBS-T, and proteins were visualized using Super Signal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL, USA). Chemiluminescence was captured using ChemiDoc XRS + System (Bio-Rad), and protein bands were quantified by densitometric analysis using Bio-Rad Molecular Imaging Software. The data are normalized with endogenous control of GAPDH and expressed in arbitrary units. All experiments were repeated three times.

For confocal analysis, cultured CAMs were grown on glass coverslips, stimulated or unstimulated, fixed in 4% paraformaldehyde in phosphate-buffer saline (PFA/PBS) for 15 min. After being permeabilized with 0.1% Triton X-100/PBS and rinsed with PBS, the cells were incubated overnight at 4°C with indicated primary antibodies: mouse anti-ubiquitin and rabbit anti-p62 (1:200; Cell Signaling, Danvers, MA, USA). After washing, the slides were probed with primary antibodies and were then incubated with Alexa-488- or Alexa-555-labelled secondary antibodies for 1 hr at room temperature. The slides were mounted and subjected to examinations by using sequential scanning on a laser scanning confocal microscope (Fluoview FV1000; Olympus), with photographs being taken and the co-localization analysed by the Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The summarized co-localization efficiency data were expressed as Pearson correlation coefficient (PCC) as described previously 19 (link).

The following antibodies were used: mouse anti-ubiquitin (Santa Cruz(P4D1), sc-8017, 1:1000); rabbit anti-LC3 (Cell Signaling Technology (D11), #3868, 1:1000); mouse anti-LAMP1 (Cell Signaling Technology (D4O1S), #15665, 1:200); mouse anti–USP8 (Sigma-Aldrich (US872), SAB4200527, 1:1000); rabbit anti-EPG5 (Beiijng TDY Biotech CO., Ltd, TDY349F, 1:1000); mouse anti-actin (Sigma-Aldrich (AC-15), A5441, 1:5000); rabbit anti-HA tag (Abcam, ab9110, 1:1000); mouse anti-FLAG tag (Abmart, M20008, 1:1000); rabbit anti-Myc tag (Abmart, M20002, 1:1000); Alexa Fluor^® 488 donkey anti-rabbit IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21206, 1:500); Alexa Fluor^® 555 donkey anti-mouse IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21203,1: 200); Alexa Fluor^® 647 donkey anti-mouse IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21235, 1:500). Chloroquine, Hoechst 33342, Baf-A1, and MG132 were obtained from Sigma-Aldrich.

Cultured CAMs were grown on glass coverslips, stimulated or left unstimulated and fixed in 4% paraformaldehyde in phosphate-buffer saline (PFA/PBS) for 15 min. After permeabilization with 0.1% Triton X-100/PBS, the cells were rinsed with PBS and incubated overnight at 4°C with indicated primary antibodies: mouse anti-vimentin (1:200, Abcam), rabbit anti-α-SMA (1:200, Abcam), rabbit anti-calponin (1:200, Abcam), rabbit anti-p62 (1:200, Abcam), mouse anti-ubiquitin (1:200, Cell Signaling) and rabbit anti-CDK1 (1:200, Cell Signaling). The slides were extensively washed and incubated with Alexa-488- or Alexa-555-labeled secondary antibodies for 1 h at room temperature. The slides were mounted and subjected to examinations by using sequentially scanning on a laser scanning confocal microscope (Fluoview FV1000, Olympus, Japan), with photos being taken, and the co-localization analyzed by the Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The summarized co-localization efficiency data was expressed as Pearson correlation coefficient (PCC) as described previously [16 (link)].