Anti ha

For co-IP and western blot analysis, 10 μg total plasmids were co-transfected into HEK293 cells, which were collected 20 h after transfection and lysed in IP buffer (1% NP-40, 50 mM Tris-HCl, pH7.4, 50 mM EDTA, 150 mM NaCl) containing protease inhibitor cocktail (Sigma). After centrifugation at 12000 rpm for 15 min at 4 °C, supernatants were collected and incubated with Protein A/G PLUS-Agarose (Santa Cruz) together with monoclonal anti-Myc, anti-Flag, or anti-HA (Abcam). After 8 h at 4 °C with soft agitation, beads were washed four times with the IP buffer above-mentioned and resuspended in 75 µl 2× SDS loading buffer. The immunoprecipitates and whole-cell lysates (WCLs) were analyzed by IB with the indicated antibodies (Abs). For western blot analysis, WCL were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) and then blotted^{64 (link),65 (link)}.
The Abs used were as follows: anti-Myc (1:1000), anti-Flag (1:1000), anti-HA (1:1000), anti-Tubulin (1:1000), and anti-mCherry (1:1000) were from Abcam. Endogenous antibodies, anti-MyD88 (1:500), anti-IRAK4 (1:500), anti-TRAF6 (1:500), anti-TAK1 (1:500), anti-calpain2a (1:500) and anti-ubiquitin (1:500) were purchased from Boster Biological Technology.

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X-100 (Solarbio, Beijing, China vT8200) for 10 min. Samples were then blocked with blocking buffer (PBS containing 10% fetal bovine serum) at 37°C for 2 h or 4°C overnight. Samples were incubated with diluted primary antibodies in blocking buffer at 37°C for 2 h or 4°C overnight. After the primary antibody incubation, samples were washed three times with PBS containing 0.1% Tween 20 (Solarbio, Beijing, China, T8220) followed by the incubation with fluorescence-conjugated secondary antibodies diluted in blocking buffer for 2 h at 37°C. Then samples were washed three times with PBS containing 0.1% Tween 20. Nuclei were counterstained with DAPI (Beyotime, Beijing, China, C1002). Images were obtained using a confocal microscope (Olympus, Tokyo, Japan, FV1200). Primary antibodies included anti-CVH (Abcam, Cambridge, United Kingdom, ab13840, 1:100), anti-CKIT (Invitrogen, CA, United States, 14-1172-81, 1:100), anti-C1EIP (polyclonal antibody, 1:10), and anti-HA (Abcam, Cambridge, United Kingdom, ab187915, 1:100). Secondary antibodies included goat anti-Rat IgG (Proteintech, Chicago, United States, SA00003-11, 1:1000, [FITC] labeled) and goat anti-mouse IgG (Proteintech, Chicago, United States, SA00003-12, 1:1000, [TRITC] labeled).