Anti flag

Immunoblotting was performed as described previously [18 (link)]. The following antibodies were used: anti-FLAG (1:1,000 dilution), anti-GFP (1:1,000 dilution), anti-β-tubulin (1:1,000 dilution), goat anti-rabbit IgG-HRP (1:2,000 dilution; Santa Cruz Inc.) and goat anti-mouse IgG-HRP (1:2,000 dilution; Santa Cruz Inc.).

The hap2Δ::HAP2-4×FLAG strain was constructed using the Gibson assembly method. The HAP2 gene in MATα was amplified using the primers listed in Table S2 and was integrated into the pNEO_4×FLAG plasmid. Integration was confirmed via enzymatic digestion and sequencing. Plasmid was linearized with the enzyme AatII for biolistic transformation into the hap2Δ (YSB1104) mutant, and reintegration into its native locus was confirmed using diagnostic PCR. To confirm the constructed strain using immunoblotting, the Hap2-4×FLAG strain was incubated in yeast extract-peptone-dextrose (YPD) broth overnight at 30°C. The overnight culture was inoculated into 50 mL of fresh YPD broth and incubated at 30°C until the optical density at 600 nm (OD₆₀₀) reached approximately 0.8. Immunoblotting with anti-FLAG (Santa Cruz Biotechnology, USA) was conducted as described previously (59 (link)).

Proteins were separated through electrophoresis in 12% SDS-PAGE gel and electro-transferred to Hybond-P PVDF membranes (Amersham Biosciences) for blotting. Immunodetection was carried out using standard methods. The anti-HA, anti-FLAG (Santa Cruz Biotechnology) and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibodies (Cell Signaling Technology) were used at a dilution of 1:1,000. HRP-conjugated secondary antibody (Sigma-Aldrich) was used at a dilution of 1:20,000. Blots were visualized with SuperSignal West Pico Chemiluminescent Substrates (Thermo Scientific) following the manufacturer’s instructions.

Human CCL2 and bovine CCL2 cDNAs were cloned into the vector pCMV-3HA (Clontech, Mountain View, CA, USA). The mature human CCL2 was cloned into pCMV-3HA after removing the N-terminal 23 amino acids of the human CCL2 precursor.
The BFV infectious clone (pBS-BFV-Z1), pCMV-3HA-BEnv, pCE-puro-3Flag-BGag, and pCE-puro-3Flag-Lck-BGag were previously described [12 (link),19 (link)]. Mutations were generated by site-directed polymerase chain reaction (PCR) (Toyobo, Osaka, Japan). All mutant plasmids were verified by DNA sequencing (Genewiz, Beijing, China) before use.
Antibodies used for protein analysis were as follows: anti-Flag, anti-HA, anti-Tubulin, and secondary antibodies labeled with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). With reference to the established laboratory protocol for preparing antibodies [37 (link)], antibodies against human CCL2 were generated in BALB/c mice using bacterially purified human CCL2 (24–99aa) protein as immunogens. Because human CCL2 (1–99 aa) forms CCL2 (24–99 aa) after being cleaved by signal protein, CCL2 antibody can be used to detect two forms of CCL2.

MG132, cycloheximide, and 3-MA were obtained from Sigma-Aldrich (St. Louis, MO, United States); anti-Myc, anti-Flag, anti-hemagglutinin (HA), anti-GAPDH, protein G agarose used for IP and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX, United States); anti-RUNX2 were obtained from Cell Signaling Technology (Beverly, MA, United States); anti-TRIM16, anti-CHIP and anti-HSP70 were from Proteintech (Rosemont, IL, United States); anti-COL1A1 was from Servicebio (Wuhan, China).

For Western blot analysis, cells were harvested and lysed with ice-cold RIPA buffer (25 mM tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Proteins from cell lysates were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Pierce, Rockford, IL, USA) using an electric transfer system. The membrane was incubated with the following antibodies: anti-ALDH1A2, anti-Flag (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Sigma–Aldrich) as a loading control. The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies, and immunoreactive bands were visualized using enhanced chemiluminescence reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The HeLa cells were co-transfected with the PC2AOE-3 × FLAG-NOC4L and pEGFP-TLR4 plasmids. After transfection for 48 h, the HeLa cells were lysed in lysis buffer containing 10 mM Tris-HCl (pH 7.8), 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 1 μM PMSF, and 1 μg/mL proteinase inhibitor cocktail (Sigma, USA). The cells were then incubated for 30 min on ice and then centrifuged at 15,000 × g for 15 min at 4 °C. The whole cell extracts (input) in the supernatant were collected and immunoprecipitated with anti-FLAG M2 magnetic beads (Sigma, USA) in accordance with the instructions provided by the manufacturer. The bound proteins were separated by SDS–PAGE, and immunoblotting was performed using anti-FLAG and anti-GFP (Santa Cruz, USA) antibodies in accordance with standard procedures.
RAW 264.7 and BMDMs cells were lysed in lysis buffer as earlier described. The whole cell extracts were immunoprecipitated with anti-NOC4L (6 R) or rIgG overnight at 4 °C and then incubated with proteinA/G (Santa Cruz, USA) for 2 h. The bound proteins were separated by SDS–PAGE, and immunoblotting was performed using anti-Noc4l in accordance with standard procedures.