Anti flag

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.

For Y2H assays, cDNA of each of the test genes was cloned into the yeast GAL4‐binding domain vector pGBKT7 and the GAL4‐activation domain vector pGADT7 (Clontech). The pGBKT7‐LAM and pGADT7 pair was used as a negative control and the pGBKT7‐53 and pGADT7 pair was used as a positive control. The pairs of Y2H plasmids were cotransformed into the yeast strain AH109. Transformants were grown on synthetic medium (SD) lacking Leu and Trp medium for 4 days, and then transferred to SD/−Ade−Leu−Trp−His medium and grown for 4 days at 30°C. At least three independent experiments were performed to confirm Y2H assay results (Liu et al., 2015).
A full‐length gene including its promoter sequence was amplified and cloned into the p1532‐GFP plasmid and pHZ‐FLAG plasmid and verified by DNA sequencing. The resultant plasmids were introduced into the protoplasts prepared from a ΔAatfb5 mutant. Transformants expressing fusion constructs were confirmed by western blotting with anti‐FLAG (Abcam) and anti‐GFP antibodies (Sigma). For Co‐IP assays, total proteins were extracted and incubated with the anti‐FLAG agarose (Sigma). Protein eluted from agarose was analysed by western blotting with anti‐GFP antibodies. The protein samples were also detected with monoclonal anti‐actin antibody (ABclonal Technology) as a control (Liu et al., 2015).