Mouse anti gapdh

The protein expression of TLR4 and NF-κB p65 in spinal cord tissue was determined using Western blotting analysis. The rats’ spinal cords were homogenized and nuclear and cytoplasmic extracts was purified from each specimen by using Nucleoprotein and cytoplasmic protein extraction kit according to the manufacturer’s instructions (KGP-150; KangChen, Shanghai, China). The antibodies used in this experiment were mouse monoclonal anti-TLR4 (Abcam), rabbit polyclonal anti–NF-κB p65 (phospho S536, Abcam), mouse monoclonal anti–Histone (Abcam) and anti-mouse GAPDH (dilution 1:10,000, Abcam) overnight on a shaker at 4°C.After three washes with TBS-0.1% Tween, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies(Bioss, Beijing, China) for 1 h. Semiquantitation of scanned images was performed using Quantity One software (Bio-Rad Laboratories, Milan, Italy).

Cell lysate and tissue sample were dissolved in RIPA buffer (Thermo) with protease inhibitor (Thermo) and phosphatase inhibitor (Thermo). Immunoblotting was performed using anti-mouse LC3B antibody (dilution 1:1000), anti- mouse beta-actin (dilution 1:1000, Sigma-Aldrich Co. Llc, SLE, U.S.A) and anti- mouse GAPDH (dilution 1:2000, Abcam).

Tissue was homogenized in CellLyticMT (Sigma) sample buffer as per the manufacturer's instructions. Protein content was quantified using the Bradford assay (BioRad). Equivalent amounts of protein were loaded onto a 10% w/v non-denaturing SDS-PAGE gel. Gels were transferred onto PVDF membrane (GE Healthcare) and incubated with the primary antibodies: rabbit anti-mouse CD248 (clone PI3 [9 (link)]) or with anti-mouse GAPDH (Abcam) followed by horseradish peroxidase-conjugated anti-rabbit IgG (Amersham, Buckinghamshire, UK). Immunodetection was carried out using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK) followed by exposure to X-ray film.

After the behavioral tests were complete, the rats were anesthetized with an overdose of pentobarbital, and the L_4–6 segments of the spinal cord were removed and processed for further biological analysis. After the samples were homogenized, cytoplasmic extracts were purified from each specimen using nucleoprotein and cytoplasmic protein extraction kits according to the manufacturer’s instructions (KGP-150; KangChen, Shanghai, China). Equivalent protein samples (50 μg) were separated using 10% SDS-PAGE, and the proteins were then transferred onto PVDF membranes (EMD Millipore, USA). The primary antibodies used in this experiment were rabbit anti-CXCL12 (1:300; Abcam, USA), rabbit anti-CXCR4 (1:200; Abcam, USA), mouse anti-TLR4 (Abcam) and anti-mouse GAPDH (dilution 1:10,000, Abcam). The membranes were incubated with the primary antibodies overnight on a shaker at 4°C. Then, the proteins were detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (BioSS, Beijing, China) for 1 h and visualized using an enhanced chemiluminescence kit (Beyotime Biotechnology, China). Semi-quantitation of the scanned images was performed using Quantity One software (Bio-Rad Laboratories, Milan, Italy).

AllStars Neg. Control siRNA (1027281) was from Qiagen and On-target plus human Notch1 siRNA smartpool was from Dharmacon (Thermo Scientific, cat# L-007771-00-0005). A total of 1.5 × 10⁶ tumor cells (tdtomato MDA-MB-231 SORE6>GFP) were transfected with 10 µl of 20 µM siRNA stock solution using Nucleofector Kit V from Lonza (cat# VCA-1003) for 48 h, as described previously^{19 (link)}. Notch1 knockdown was confirmed using western blotting with the following antibodies: rabbit anti-Notch1 at 1 : 1000 (Cell Signaling, cat# 3608), rabbit anti-Notch2 at 1 : 1000 (Cell Signaling, cat# 5732), and mouse anti-GAPDH at 1 : 10,000 (Abcam, cat# ab8245). At 36 h post siRNA transfection, tumor cells were plated with macrophages (1 : 5 ratio) overnight. Next day, cells were imaged live on DeltaVision microscope as described above.

Sodium arsenite (Sigma-Aldrich) was added to the cells at a concentration of 250 μM for 20 min prior to fixation or cell lysate collection. The PKR inhibitor C16 (Sigma-Aldrich) was added to infected cells at a concentration of 1 μM at 1 hpi, and cell lysates were collected at 12 hpi. The ISR inhibitor ISRIB (Sigma-Aldrich) was added at 1 hpi at 0.5 μM. Puromycin (Life Technologies) was added to cells at a concentration of 10 μg/ml at indicated times prior to cell lysate collection. Poly(I⋅C) (Sigma-Aldrich, catalog no. P1530) is a dsRNA analogue and was used as a positive control for immune activation. Poly(I⋅C) was used at a concentration of 20 μg/ml and added to the cell culture medium in combination with Lipofectamine 2000. MNV ORF1 plasmids were described and used previously (22 (link)).
Goat anti-eIF3η, goat anti-G3BP1, and goat anti-TIA-1 were all purchased from Santa Cruz Biotech. Rabbit anti-eIF2α was purchased from Invitrogen; Rabbit anti-actin from Sigma-Aldrich; Mouse anti-Puromycin was obtained from Kerafast, Inc. Mouse anti-G3BP1, mouse anti-GAPDH, rabbit anti-His, and rabbit anti-calnexin were obtained from Abcam, and rabbit anti-p-eIF2α (S52) and Alexa Fluor-conjugated species-specific IgG were purchased from Life Technologies. Rabbit anti-NS7 and rabbit anti-NS5 were manufactured and produced by Invitrogen.